免疫组化

免疫组化 免疫组化基础知识 admin 免疫组织化学的概念: 免疫组化是利用抗原与抗体特异性结合的原理，通过化学反应使标记抗体的显色剂 (荧光素、酶、金属离子、同位素) 显色来确定组织细胞内抗原(多肽和蛋白质)，对其进行定位、定性及定量的研究，称为免疫组织化学。 免疫组化实验所用的抗体有哪些, 免疫组化实验中常用的抗体为单克隆抗体和多克隆抗体。单克隆抗体是一个B淋巴细胞克隆分泌的抗体，应用细胞融合杂交瘤技术免疫动物制备。多克隆抗体是将纯化后的抗原直接免疫动物后，从动物血中所获得的免疫血清，是多个B淋巴细胞克隆所产生的抗体混合物。 免疫组化实验所用的组织和细胞标本有哪些, 实验所用主要为组织标本和细胞标本两大类，前者包括石蜡切片(病理大片和组织芯片)和冰冻切片，后者包括组织印片、细胞爬片和细胞涂片。 其中石蜡切片是制作组织标本最常用、最基本的方法，对于组织形态保存好，且能作连续切片，有利于各种染色对照观察;还能长期存档，供回顾性研究;石蜡切片制作过程对组织内抗原暴露有一定的影响，但可进行抗原修复，是免疫组化中首选的组织标本制作方法。 石蜡切片为什么要做抗原修复,有哪些方法, 石蜡切片标本均用甲醛固定，使得细胞内抗原形成醛键、羧甲键而被封闭了部分抗原决定簇，同时蛋白之间发生交联而使抗原决定簇隐蔽。所以要求在进行IHC染色时，需要先进行抗原修复或暴露，即将固定时分子之间所形成的交联破坏，而恢复抗原的原有空间形态。 常用的抗原修复方法有微波修复法，高压加热法，酶消化法，水煮加热法等，常用的修复液是pH6.0的0.01 mol/L的柠檬酸盐缓冲液。 免疫组化常用的染色方法有哪些, 根据标记物的不同分为免疫荧光法，免疫酶标法，亲和组织化学法，后者是以一种物质对某种组织成分具有高度亲合力为基础的检测方法。这种方法敏感性更高，有利于微量抗原(抗体)在细胞或亚细胞水平的定位，其中生物素——抗生物素染色法最常用。 抗体交叉反应的原因: 指抗体除与其相应的抗原发生特异性反应外还与其它抗原发生反应。产生的原因有以下几个方面: 1. 抗原特异性指用于免疫动物的抗原性物质中含有多种抗原分子，它引起动物产生针对多种抗原分子特异性的相应抗体。任何其它物质只要含有一种或多种与上述物质相同的抗原分子，必将与上述多特异性的抗血清发生交叉反应。 2. 共同决定簇即两种抗原分子中都含有相同的抗原决定簇。 3. 决定簇相似，两种不同的抗原决定簇，如果结构大致相同，由于空间构象关系，某一决定簇的相应抗体可以与大致相同的决定簇发生交叉反应。当然抗原一抗体之间构象相似时的结合力小于吻合时的结合力。 免疫细胞化学技术 一、免疫细胞化学技术的概述 *免疫细胞化学(immunocytochemistry, ICC) -是利用抗原与抗体特异性结合的原理，通过化学反应使标记抗体的显色剂 (荧光素、酶、金属离子、同位素) 显色来确定细胞内抗原的成分(主要是多肽和蛋白质)，对其进行定位、定性及定量的研究，称为,。 (一) 对抗原和抗体的要求 *具有特异性高和亲和力强的抗体是实验成功的首要条件。 -对抗体的要求:纯度高、比活性强; *高度特异性抗体的获得，取决于抗原的纯度。 -对抗原的要求:纯度高，免疫原性强，稳定无变化。 (二) 抗原 (antigen, Ag) *抗原的概念:凡是在机体内引起体液免疫和(或)细胞免疫反应的物质，称为抗原。抗原具有两个方面的特性: -免疫原性:引起机体产生抗体和(或)致敏淋细胞的特性; -免疫反应性:抗原能与相应的抗体及致敏淋巴细胞发生特异的结合或反应的特性。 *根据抗原是否显示免疫原性分为: -完全抗原:分子量较大，一般在10kDa以上，并具有较复杂的化学组成。 *免疫原性最强的是蛋白质抗原，多糖次之;脂类和核酸必需和蛋白质及多糖形成复合物才具有良好的免疫原性。 -半抗原:又称为不完全抗原，分子量较小。例如:某些短肽、多糖、类脂和药物等。 *半抗原必需与载体结合，才能获得免疫原性。 载 体 *通常是具有高度免疫原性的大分子物质，具有将免疫原性传递给耦联的半抗原能力。 -常用的载体有钥孔血蓝蛋白(keyhole limpet hemocyanin，KLH)、牛血清白蛋白(bovine serum albumin, BSA)、卵白蛋白(Ovalbumin，OVA)等。 -用戊二醛或碳化二亚胺作为交联剂通过功能基团-NH2、-COOH等将半抗原结合到载体上。结合比例为5kDa结合5,25个分子的小肽。 (三)抗体 (antibody, Ab) 1、抗体的概念: *机体受到抗原刺激后，由浆细胞合成并分泌出一类具有与抗原发生特异性结合的球蛋白，被称为抗体。 -抗体主要存在于血清内; -抗体都是免疫球蛋白，但免疫球蛋白并不一定都是抗体。 -免疫球蛋白根据重链的结构及抗原特异性不同分为五种，既IgG、 IgD、IgE、 IgA、IgM。 2、免疫组化实验中常用的抗体:单克隆抗体和多克隆抗体。 *单克隆抗体:是一个B淋巴细胞克隆分泌的抗体，是应用细胞融合杂交瘤技术免疫动物制备的。 -特异性强、抗体产量高。 *多克隆抗体:是将纯化后的抗原直接免疫动物后，从动物血中所获得的免疫血清，是多个B淋巴细胞克隆所产生的抗体混合物。 -特异性低，会产生抗体的交叉反应。 -多克隆抗体广泛应用于石蜡包埋组织切片，可减少假阴性染色机会。 -抗原与抗体的结合，要求量保持一定比例，当抗原或抗体过量时，是不能聚合成大颗粒。 3、抗体的制备——多克隆抗体的制备 *动物的选择 *佐剂(adjuvant) *免疫方法 *免疫剂量 *抗体效价的测定 *放血或定期抽血 (1) 动物的选择 选择什么动物来免疫取决于: *所需抗血清的量 -小鼠只能提供1.0,1.5ml的血液，而山羊能提供几升。 *能供免疫用的抗原量 -小鼠足够，而山羊需要几毫克。 (1) 动物的选择(续) *动物的品系:免疫动物与提供抗原的动物之间的种系差异越大越好。 -例如哺乳动物的抗原可选择非哺乳动物来制备抗体。 -常用的动物有兔、羊、马、猪等，以兔(新西兰兔，年轻，健壮，体重在2.5kg左右，雄性)为最常用。 (2) 佐剂 (adjuvant) *一般可溶性抗原注射后，迅速从注射部位扩散、吸收代谢。佐剂可延长潴留时间，且延长免疫刺激作用。因此在制备抗体的过程中，常将抗原和佐剂一起注射。 *最常用的佐剂是弗氏佐剂，又分为: -弗氏不完全佐剂(Freund's incomplete adjuvant) -弗氏完全佐剂(Freund's complete adjuvant) 弗氏不完全佐剂 (Freund's incomplete adjuvant, FIA) *是由油剂(石蜡油或植物油)与乳化剂(羊毛脂或Tween80)混合而成，比例为1:1、2:1、3:1或5:1，可根据需要而定，通常2:1。 *使用时与水溶性抗原按1:1比例充分混合，使抗原分散在佐剂中形成油包水乳剂。 弗氏完全佐剂 (Freund's complete adjuvant, FCA) *在弗氏不完全佐剂中加入活卡介苗或死的结核分枝杆菌(终浓度为2,20mg/ml)，即成为FCA。 *免疫动物时，将弗氏佐剂与抗原按体积 1:1 混合乳化后(油包水)注入动物。 -一般首次注射时用完全佐剂乳化，第二次或第三次注射时用不完全佐剂或不用佐剂。 佐剂与抗原乳化的方法 *研磨法:适于制备大量的佐剂抗原 -先将不完全佐剂加热，取1.73ml放人无菌玻璃研钵内;缓缓滴入0.23ml活卡介苗，边滴边按同一方向研磨，使菌体完全分散。 -按同样方法滴人抗原，每加一滴应研磨至小滴消失。滴加抗原的速度要慢，待抗原全部加人后，应成为乳白色粘稠的油包水乳剂。 *缺点:研钵壁上粘附大量乳剂，抗原损失较大，对微量或难得抗原不宜采用。 佐剂与抗原乳化的方法(续) *注射器混合法 -将等量的完全佐剂和抗原分别吸人两个5ml注射器内，然后插入三通管内，交替推动针管 混匀，往复操作直至形成粘稠的乳剂为止。 *优点:无菌操作，节省抗原或佐剂。 *缺点:不易乳化，时间长。 佐剂与抗原乳化的方法(续) *快速乳化法:利用超声波粉碎器可快速乳化抗原和佐剂混合物。 -将抗原和佐剂按所需量加入一离心管中，置于超声波粉碎器上，粉碎头浸入液面下 0.5cm，离瓶底0.5cm左右，以免打碎离心管。 -每次乳化 10,15s，然后置冰箱lmin左右。反复乳化3,4次即可完全乳化。管内残余量800r/min离心5,10min收集。 *优点:简单、快速、节省。 乳化剂的鉴定 *判断乳化是否充分，可将一滴乳化好的液体滴在水面上(冷水中)，如能长时间保持圆珠形而不散开，示乳化达到要求。 (3) 免疫方法——免疫途径 *常有静脉、腹腔、肌肉、皮下、皮内、淋巴结、脚掌等注射。 *一般采用多点注射方法 -常在足、掌、腋窝淋巴结周围、背部两侧、颌下、耳后等处的皮内或皮下。 -皮内易引起细胞免疫反应，有利于提高抗体的效价。 (3) 免疫方法——免疫途径 (续) *几点说明: -大动物一般不用腹腔注射 -颗粒抗原和使用佐剂时不能静脉注射 -抗原宝贵可采用淋巴结内微量注射法，只需10,抗原即可获得较好的免疫效果。 -皮内注射较困难，特别是天冷时更难注入。 (3) 免疫方法——次数及间隔时间 *次数一般为2,3次(初次免疫和加强免疫) -首次注射后，10,15天再加强注射; -剂量同首次或为首次的一半，用不全佐剂或不用佐剂。 *间隔时间，一般而言，动物越大，间隔越长 -豚鼠、大鼠为7,8天，兔子为10,15天，羊为14,28天; -第三次注射的间隔时间更长些，效果更好。 举例1:家兔的免疫 *初次免疫 -用50,加入FCA，在背部皮下注射6,8点，每点0.1,0.2ml，也可肌肉内或皮内注射; *两周后，加强免疫 -将50,于PBS或FIA中，在肌肉、皮下、静脉或腹腔内注射; *抗体效价的检测:加强免疫一周后，耳缘静脉采血 -抗体效价测定可用环状沉淀试验、琼脂双向扩散法等方法。前者抗体效价在1:4000以上，后者在1:16以上，即可采血，取血清进一步提纯。 举例2:微量抗原淋巴结内注射免疫家兔 *一般选择2.5,3.0kg的新西兰种公兔 -先在其两脚垫注射完全福氏佐剂0.2ml(卡介苗每兔合5mg); -10天后，见胭窝淋巴结肿大，然后将抗原注射至肿大的淋巴结内，第一次注射抗原用完全 福氏佐剂混合乳化; -以后每隔20天用不完全福氏佐剂乳化抗原，以同样方法加强2次，各次注射的抗原均为25,; -末次注射两周后兔耳放血测价 (可达1:128)。 举例3:豚鼠和大鼠的免疫 *初次免疫 -用10,加入FCA，在背部皮内注射4,6点，每点 0.lml，也可肌肉内或皮下注射; *加强免疫 -每隔 7,8天，将10,于 PBS或FIA中。在肌肉、皮下或静脉注射; *抗体效价的检测 (4) 免疫剂量 *抗原性强的抗原量应小，过大反会引起免疫抑制; *免疫周期长的动物可少量多次注射，短的可大量少次。 (4) 免疫剂量 (续) *各种动物首次免疫抗原剂量和加强免疫的剂量 (5) 抗体效价的检测 多采免疫双向扩散法 【基本原理】 *指抗原和抗体在同一凝胶内都扩散，彼此相遇后形成特异性的沉淀线。 -将抗原与抗体分别加入同一凝胶板中两个相隔一定间距的小孔内，使两者进行相互扩散，当抗原抗体浓度之比相适宜时，彼此相遇形成一白色弧状沉淀线。 *优缺点:简便，但敏感性较低，易出现假阴性结果。 免疫双向扩散法的操作步骤 *将玻璃板用水洗净后用75,乙醇冲洗，晾干后放在水平台上备用。 *将1,agar 融化后，置56?水浴。 *在玻璃板上铺胶，约1.5mm 厚，凝固后打孔(直径 3rnm)，孔间距10mm。 *中心孔加抗原样品，周围孔内每孔加抗血清(抗体预先作系列倍比稀释即 1:2、1:4、1:8、1:16、1:32等比例)。 *凝胶板置湿盒内，室温扩散24h。 *观察结果。 抗体效价检测的其它方法 *环状沉淀试验，需较多的抗血清，现已很少用; *对流免疫电泳，比琼脂免疫双扩散法敏感，较简便、实用; *酶联免疫吸附试验 (ELISA)。 放血或定期抽血 常用以下3种方法 *颈动脉放血法:放血量较多，动物不易中途死亡。例如:2.5kg白兔可放血约80ml。家兔、山羊、绵羊等动物采血常用此法。 *心脏采血法:常用于家兔、豚鼠、大白鼠、鸡等小动物，但操作不当易引起动物死亡。 *静脉采血:家兔可用耳缘静脉采血;山羊、绵羊、马和驴可用颈静脉采血，这种放血法可隔日1次，有时可采集多量血液。 放血或定期抽血 (续) *采血过程中，动作要轻柔，尽量避免溶血 *血液凝固后，及时离心收集血清，否则细胞溶解释放的杂蛋白(例如蛋白水解酶)将污染抗体并将抗体水解，降低效价; *加叠氮化钠，分装，低温保存，也可加一定的保护剂如BSA、甘油等。 二、组织和细胞标本的制备 *标本制备恰当，是免疫组化成功的首要条件 *免疫组化对组织和细胞标本的要求 -保持所检标本原有的结构、形态; -在原位最大程度地保持待测抗原(或抗体)的免疫活性，既不淬灭、流失或弥散，也不被隐蔽。 *免疫组化的组织和细胞标本，制作流程与常规处理方法基本相同，但对组织、细胞的处理又有其特殊要求及注意事项。 -各种抗原由于其含量及特性的差异对标本处理方式常有不同要求，因此要选择适用于本实验的最佳方法。 免疫组化中常用的组织和细胞标本 *组织标本 -石蜡切片 -冰冻切片 *细胞标本 -组织印片 -细胞培养片(细胞爬片) -细胞涂片 (一) 石蜡切片 *石蜡切片是制作组织标本最常用、最基本的方法 -最大优点是组织形态保存好，且能作连续切片，有利于各种染色对照观察; -石蜡块还能长期存档，供回顾研究。 *石蜡切片制作过程对组织内抗原显现有一定的影响，但可通过某些措施予以改善，因而它是大多数免疫组化中首选的组织标本制作方法。 1、取材的特殊要求及注意事项 *标本新鲜:一般在2h以内进行，超过2h，组织将有不同程度的自溶，其抗原或变性消失，或严重弥散。 *取材部位:除取病灶或含待检抗原部位外，还应取病灶与正常交界处，即所取组织切片中同时应有抗原阳性和阴性区，以形成自身对照。 -细胞坏死后，不仅抗原弥散或消失，而且常引起非特异着色，干扰观察，因此取材时应尽可能避开坏死区。 1、取材的特殊要求及注意事项(续) *避免挤压:取材时组织受挤压可使边缘部细胞形态改变并加深非特异着色，因而取材时应使用锋利的刀刃; -镊取组织动作要轻; -经窥镜直接钳取的组织往往有过度挤压，观察结果时应有所考虑。 2、固定及常用的固定液 *取材后的组织需立刻投于固定剂中 -固定使组织和细胞的蛋白质凝固，终止内源性或外源性酶反应，防止组织自溶或异溶，以保持原有结构和形态; -对免疫组化而言更有原位保存抗原的作用，避免抗原失活或弥散。 *常用固定液:固定剂品种很多，但大多属于醛类和醇类。其固定原理不同，各有优缺点。 -醛类(常用甲醛、戊二醛和多聚甲醛) -醇类(常用乙醇) -其它 (丙酮) (1) 醛类 *甲醛(福尔马林)应用最广 -原理:形成分子间的交联，影响蛋白构型而使之固定。 -优点:形态结构保存好，且穿透性强，组织收缩少。 -缺点: *甲醛放置过久可氧化为甲酸，使溶液pH降低，影响染色; *醛基与抗原蛋白的氨基交联形成羧甲基，使抗原决定簇的三维构象出现空间障碍; *分子间交联形成的网格结构可能部分或完全掩盖某些抗原决定簇，使之不能充分暴露。可造成假阴性的染色结果。 -注意事项: *缩短固定时间，降低固定温度，为此组织块不宜过厚。 *改用中性缓冲福尔马林，以pH值7.2,7.4 0.01mol/L的磷酸盐缓冲液配制成10,甲醛固定液，减少固定液pH的变化。 *固定后充分水洗以减少分子间交联。 *切片在作免疫组化染色前，先经预处理使抗原再现(抗原修复)。 *戊二醛: -穿透性强，微细结构保存好，但对抗原有一定影响，常与其他固定剂联合用作免疫电镜固定液。 *多聚甲醛(常用4%): -可用于免疫电镜;也可用于免疫荧光染色。 *主要检测组织内一些性能娇弱的抗原特别是细胞表面抗原，例如各类淋巴细胸分化决定簇(CD)、主要组织相容性抗原等。 (2) 醇类 * 最常用的醇类固定剂是乙醇。 -其固定作用:使细胞内蛋白、糖类发生沉淀。 -优点:穿透性强、抗原性保存好。 -缺点: *脱水性强，易引起组织收缩、变硬，影响切片质量，因而乙醇固定时间不宜过长(2h内)。 *乙醇使蛋白变性的作用轻，固定后可再溶解;染色过程中，温育时间长，抗原可流失而减弱反应强度。 (3) 其它固定剂 *丙酮: -对抗原性的保存好，但脱水性更强，较少 用于组织标本，但细胞爬片常用丙酮固定。 3、抗原修复——原因 *常规的石蜡切片标本均用甲醛固定，结果使得: -抗原性物质形成醛键、羧甲键而被封闭了部分抗原决定簇; -蛋白之间发生交联而使抗原决定簇隐蔽。 *要求:在染色时，需要先进行抗原修复或暴露，即将固定时分子之间所形成的交联破坏，而恢复抗原的原有空间形态。 3、抗原修复——方法 *化学方法 *加热方法 -水浴加热法 -微波照射法 -高压加热法 -酸水解法 (1) 化学方法 *主要是通过一些酶的作用，使抗原决定簇暴露。常用的酶有:胰蛋白酶、胃蛋白酶等。 -胰蛋白酶:一般使用浓度为0.05,,0.1,，消化时间为，10,40min，主要用于细胞内抗原的显示; -胃蛋白酶:一般使用浓度为0.1%,0.4,，消化时间为、30,180min，主要用于细胞间质抗原的显示。 *例如:Laminin、CollagenIV (2) 水浴加热法 *将玻片放入装有抗原修复液的容器中，加热至沸腾，持续10,15分钟。 -优点:操作简单、经济，适用于所有的实验室， -缺点:对封闭牢固的抗原决定簇暴露不理想。 (3) 微波照射法 *将玻片放入装有抗原修复液的容器中，置微波炉加热至95?以上，持续l0,15分钟，冷却后，按免疫组化染色步骤进行。 *此方法由于微波场内极性分子、离子高速运动，撞击交联的网链，使抗原异常的构想恢复正常，且因分子运动产热、效率高、时间短，对抗原再现效果好。 (4) 高压加热法暴露抗原 *将玻片浸入抗原修复液内，置高压锅中高压2,3min，可取得极好的效果。 *由于高压下受热均匀，特别使用于大批量标本的染色。 (1) 酸水解法 *酸水解可使交联断裂、暴露抗原。 *将玻片浸入1mol/L HCl 溶液中，室温作用20min(温度升高，作用时间缩短)。 *此法能增强特异性染色，降低背景，但需注意水解过度将破坏抗原性及形态结构。 加热法的注意事项 *达到规定的温度(92,以上); *维持一定的时间; *避免切片干涸 (抗原可能完全丢失); *加热后必须经过室温自然冷却20,30分钟，使未折叠的蛋白分子链恢复天然构型; *修复液: -最常用的是pH6.0的0.01mol/L的枸橼酸盐缓冲液; -最新研究表明碱性修复液更有效，推荐使用1mM的EDTA缓冲液(pH8.0)。 4、载玻片的处理 *抗原修复过程中，由于高温、高压等诸多固素的影响，极易造成脱片。为防止脱片，常用粘附剂处理载玻片。 -新载玻片上有油污，要用洗液浸泡12,24h，自来水冲洗后再用蒸馏水清洗;用绸布擦干或烤箱烤干。清洁的载玻片再用粘附剂处理。 *常用的粘附剂有: -APES(3-氨丙基三乙氧基硅烷) -Polv-L-Lysine(多聚赖氨酸) -铬明胶溶液 APES(3-aminopropyltriethoxysilane) 3-氨丙基三乙氧基硅烷 *现用现配。用纯丙酮或甲醇配制2%APES(v/v); *将洗净的玻片浸于此液中20,30秒钟; *取出稍停片刻，再入纯丙酮酮溶液洗去未结合的APES，置通风橱中晾干或烤箱烤干。 Polv-L-Lysine (多聚赖氨酸) *将清洁玻片浸于的多聚赖氨酸溶液中(去离子水稀释)，放置30min，然后烤箱烘烤1h或室温过夜干燥。装盒备用。 铬明胶溶液 *试剂:铬明矾(chrome alum) 0.25g 明胶(gelatine) 2.5g 蒸馏水 500ml *先将铬明矾溶解于少量蒸馏水中，再加入明胶及蒸馏水，可在水浴中使明胶溶化，搅拌均匀后，即可使用。如有残渣，可过滤后再用。 *用时稍加溶化，切片浸入2,3min，过夜晾干。 (二) 冰冻切片 *冰冻切片是指将组织在冷冻状态下直接切片。 *在切片前组织不经过任何化学药品处理或加热过程。 -缩短了制片时间 -抗原性不受损失 *对稳定性差的抗原，如淋巴细胞表面抗原尤其适合。 *组织冻结过程中，细胞内、外的水分会形成冰晶，冻结的速度愈慢，冰晶颗粒愈大，可严重影响组织、细胞的形态结构。因此制备冻块时要求低温、速冻。 1、冰冻组织块的常用方法 *液氮中冰冻:组织投入液氮中 (一中10,20sec; *干冰中加入丙酮(或异戊烷)，液体立即气化起泡，温度降至一，将组织投入，若在干冰丙酮中置一盛有异戊烷的容器，组织投入该容器内结冻则更好; -上述组织在速冻时应浸埋于OCT包埋剂或甲基纤维素糊状液内，以保护组织。 -制成冻块后若需保存，应以铝箔或塑料薄膜封包，贮存于-冰箱。 2、切片 *供免疫组化用的冰冻切片同样要求附贴平整，并有连续性。 *载玻片也应清洁无油污，但一般无需涂抹粘附剂; *切片时，使用恒温冷冻切片机，在箱内温度-。切片厚度一般为4,。 3、切片后处理 *切好的冰冻切片，室温下自然晾干1,2h后，入丙酮固定10min，待干燥后作免疫组化染色或封存于-20?。 *冰冻切片由于切片技术要求较高，不易得到连续性很好的切片，其形态结构亦不如石蜡片，且冻块和切片不便于长期贮存，因此冰冻切片的应用受限。 (三) 组织印片 *将洁净载玻片轻压于已暴露病灶的新鲜组织切面，细胞即粘附于玻片，晾干后浸入冷丙酮或醋酸-乙醇固定10min，自然干燥后染片或-20?保存。 (四) 细胞培养片(细胞爬片) *贴壁细胞培养时，置盖片于培养瓶中，使细胞在盖片上生长，达适当密度后取出固定(丙酮-固定10,20min)，再进行免疫染色。 -盖片的处理方法同载玻片的处理，但泡酸时间2h即可。 -为了防止细胞脱片，可用多聚赖氨酸处理。 (五) 细胞涂片 *大多数细胞涂片由细胞悬液制成，包括: -血液、尿液、脑脊液; -体腔积液; -组织穿刺吸取，如骨髓、淋巴结或其他实质性组织 -悬浮培养的细胞或贴壁细胞经消化后形成的悬液。 (五) 细胞涂片 (续) *细胞涂片的方法: -手涂法 *将细胞浓度调节到106/ml左右，可直接涂于载玻片上，但要均匀、不重叠。 *图片范围应小于1cm直径，以节约试剂。 -涂片机涂片法 *将细胞样品制成2×105,6/ml 细胞悬液，吸取50,,2×104,5cells) 加入涂片机内，1000rpm离心2min后细胞就均匀分布于玻片上。 三、免疫组化常用的染色方法 *根据标记物的不同分为 -(一) 免疫荧光法 -(二) 免疫酶标法 -(三) 亲和组织化学法 (一) 免疫荧光法 【原理】 *用于免疫荧光的标记物是小分子的荧光素，可标记抗体或抗原; *荧光素经某种特定波长的光照射激发后，能发射出一种比激发光波波长更长而且能量较低的荧光，籍此可作定位观察或示踪; *借助于荧光显微镜进行观察。 1、常用的荧光素 *(1) 异硫氰酸荧光素 (Fluorescein Isothiocyanate, FITC) *(2) 四甲基异硫氰酸罗丹明 (Tetramethyl Rhodamine Isothiocyanate, TRITC) *(3) 四乙基罗丹明 (RB200) *(4) 碘化丙啶 (propidium iodide, PI) (1) 异硫氰酸荧光素(FITC) *易溶于水和乙醇。 *最大吸收光谱为490,495nm，最大发射光谱为 520,530nm(呈翠绿色荧光，分子量 389.4。 *在碱性条件下，FITC的异硫氰酸基在水溶液中与Ig的自由氨基形成共价键，成为标记的荧光抗体。一个lgG分子上最多能标记15,20个FITC分子。 (2) 四甲基异硫氰酸罗丹明(TRITC) *最大吸收光谱550nm，最大发射光谱620nm，呈红色荧光，分子量为444。 *与蛋白质结合的方式同FITC。 (3) 四乙基罗丹明(RB200) *不溶于水，易溶于乙醇和丙酮。 *最大吸收光谱为570nm，最大发射光谱为595,600nm，呈橙红色荧光，分子量为580。 *RB200在五氯化磷(PCl5)作用下转变成磺酰氯(SO2Cl)，在碱性条件下，易与蛋白质的赖氨酸-氨基反应而标记在蛋白分子上。 (4) 碘化丙啶(PI) *是常用的DNA荧光标记探针，可作为FITC的胞核对比染色。 *PI可嵌入到双链DNA和RNA碱基对中并与之结合，但对碱基无特异性选择。 *最大吸收光谱是493nm，最大发射光谱是630nm，呈红色荧光。 2、荧光抗体的保存 *一要防止抗体失活，二要保持荧光素不脱落和不受激发猝灭。 -一般认为0,可保存1,2年，-可保存3,4年。 -要小量分装，防止反复冻融。 -保存前需加防腐剂 (浓度为1:5000,10000的硫柳汞或1:1000,5000叠氮化钠) 。 3、免疫荧光的染色方法 *免疫荧光染色法常用的有 -直接法 -间接法 ?原理将荧光素标记在相应的抗体上，直接与相应抗原反应 (用来检测未知抗原)。 ?直接免疫荧光法的操作步骤 *标本的处理: -细胞涂片、细胞爬片浸入冷丙酮或4%的多聚甲醛固定10min，然后用0.0lM PBST (含0.l%TritonX-100 pH 7.4) 漂洗5min × 3/次; -石蜡切片经脱蜡、梯度酒精脱水后，进行抗原修复，然后用0.01M PBST漂洗5min × 3/次; *2%BSA或湿盒内封闭30min *抗体染色: -在标本片上滴加适当稀释的荧光标记抗体(1:8或1:16稀释)，放在湿盒中，孵育30min; 直接免疫荧光法的操作步骤(续) *0.0lmol/L PBS(pH 7.4) 漂洗5min × 3/次，不时震荡(洗去多余游离的荧光素标记的抗体)。 *缓冲甘油封片 -纯无荧光的甘油9份+ pH 9.2，0.2M碳酸盐缓冲液1份配制。 *镜检:在荧光显微镜下观察。 *优点:方法简便、特异性高，非特异性荧光染色少。 *缺点:敏感性偏低;而且每检查一种抗原就需要制备一种荧光抗体。若检测多种抗原需制备多种相应的荧光标记抗体。 直接免疫荧光法的注意事项 *对荧光标记的抗体的稀释:要保证抗体的蛋白有一定的浓度; *一般稀释度不应超过1:20，抗体浓度过低，会导致产生的荧光过弱，影响结果的观察。 直接免疫荧光法的注意事项 (续) *染色温度和时间需要根据各种不同的标本及抗原而变化; -染色时间:从10 min到数小时，一般30 min; -染色温度:多采用室温，高于可加强染色效果，但对不耐热的抗原(如流行性乙型脑炎病毒)可采用0-的低温，延长染色时间。 -低温染色过夜较效果好的多。 直接免疫荧光法的注意事项 (续) *试验时需设置下列对照: -自发荧光对照(空白对照):标本加0.01mol/L，pH7.4的PBS代替一抗。 -阳性对照:用已知的阳性标本加荧光标记的特异性抗体。 -特异性对照(抑制试验):标本加未标记的特异性抗体，再加荧光标记的特异性抗体。 *若标本自发荧光对照和特异性对照呈无荧光或弱荧光，阳性对照和待检标本呈强荧光，则为特异性阳性染色。 直接免疫荧光法的注意事项 (续) *一般标本在高压汞灯下照射超过3min，就有荧光减弱现象; *经荧光染色的标本最好在当天观察，随着时间的延长，荧光强度会逐渐下降。 (2) 间接法又称为荧光抗-抗体法 需要两种抗体参与，即一抗和二抗(荧光素标记)。一抗对标本中的抗原来说起抗体的作用，但对荧光标记的二抗来说又起着抗原作用。 -可用来检测标本中未知抗原，也可检测血清中未知抗体。 间接免疫荧光法操作步骤 *标本的处理及非特异染色的封闭同直接法; *一抗染色: -加未标记的特异性抗体(通常1:100稀释，用0.01MpH7.4的PBS稀释)，作用30min或过夜。 *0.01M PBST漂洗5min×3次(震荡漂洗); 间接免疫荧光法操作步骤(续) * 加荧光标记的二抗抗体，湿盒避光作用30min。 *0.01M PBST避光漂洗5min×3次(例如包上锡纸，在摇床上漂洗); *甘油缓冲液封片 *镜检 *优点:敏感性较高，比直接法高10倍左右;制备一种荧光标记抗体，可应用于多种一抗; *缺点:是参加反应的因子较多，产生非特异性染色的机会增多。 间接免疫荧光法的注意事项 *荧光染色后一般在1h内完成观察，或于4?保存4h，时间过长 ，会使荧光减弱。 *每次试验时 ，需设置以下三种对照: -阳性对照:阳性血清+荧光标记物 -阴性对照:阴性血清+荧光标记物 -荧光标记物对照:PBS+荧光标记物 间接免疫荧光法的注意事项(续) *标本片需在操作的各个步骤中，始终保持湿润，避免干燥。 *一抗和二抗应始终保持在标本片上，避免因放置不平使液体流失，从而造成非特异性荧光染色。 (二) 免疫酶酶标法 【原理】 *以酶作为标记物与外加底物作用后产生不溶性色素，沉积于抗原和抗体反应的部位; *酶降解底物的量与色泽浓度成正比。可反映被测定的抗原或抗体的量。 1、常用的标记酶及其显色底物 *辣根过氧化物酶(horseradish peroxidase, HRP)及底物 *碱性磷酸酶(alkaline phosphatase, AP)及底物 (1) 辣根过氧化物酶(HRP)及底物 *HRP是应用最广的一种酶，来源于植物辣根，由无色的酶蛋白和深棕色的铁叶琳结合而成，分子量约40kDa，稳定性好; *底物为过氧化物和供氢体(DH2) -过氧化物:常用过氧化氢和过氧化氢尿素。 -供氢体:多用无色的还原型染料，通过反应生成有色的氧化 型染料，最常用的供氢体是DAB。 DAB (二氨基联苯胺) *DAB本身无色，反应后呈棕色，不溶于水，不易褪色，电子密度高，最为常用。 *目前已经有商品化的试剂盒，使用起来非常方便。 (2) 碱性磷酸酶(AP)及底物 *AP为磷酸酯的水解酶，可通过两种反应显色: -偶氮偶联反应，底物为-萘酚磷酸盐，经水解后得-萘酚，与重氮化合物如坚牢蓝(fast blue)或坚牢红(fast red)形成不溶性沉淀，分别呈兰色或红色。 -靛蓝-四唑反应:底物为溴氯羟吲哚磷酸盐(5-bromo-4-chloro-3-indodyl phosphate,BCIP)，经酶水解并氧化形成靛蓝，而氮蓝四唑(NBT)在此氧化过程中被还原成不溶性紫兰色沉淀。 2、常用的免疫酶染色方法 *又分为以下两种方法: -酶标抗体法 *直接法 *间接法 -非标记抗体酶法 *酶桥法 *PAP法 (1) 酶标抗体法 *通过共价键将酶结合在抗体上，制成酶标抗体，与标本进行反应后，再用酶组化法将酶显色，使之生成有色的不溶性产物或具有一定电子密度的颗粒，以供光镜和电镜观察。 -优点:切片能长期保存、反复观察，适于镜下半定量分析。 -缺点:酶与抗体形成的共价键，可损害抗体和酶的活性;易产生非特异染色。 (1) 酶标抗体法——直接法 • 将酶直接标记在一抗上，然后直接与相应抗原特异地结合。形成抗原-抗体-酶复合物，最后用底物显色剂显色。 (1) 酶标抗体法——间接法 *将酶标记在二抗上，先将一抗与相应的抗原结合，形成抗原抗体复合物，再用二抗(酶标抗体)与复合物中的特异抗体结合，形成抗原-抗体-酶标抗体复合物，最后用底物显色剂显色。 酶标抗体间接法的操作步骤 *标本准备: -石蜡脱蜡至水; -冰冻切片浸入丙酮固定10min，0.01M PBST漂洗，5min×3; -细胞爬片先用PBS洗，然后丙酮固定10min，再0.01M PBST漂洗，5min×3; *石蜡切片需要进行抗原修复，其它标本则不用; (2) 酶标抗体间接法的操作步骤(续) *封闭内源性过氧化物酶:3,H2O2-甲醇溶液室温孵育5,10min (湿盒内) ; *0.01M PBST漂洗，5min×3; *5,10%正常山羊血清(0.01M PBS稀释)封闭，室温孵育30min (湿盒内) ; *倾去血清勿洗，加1,BSA(PBST配制)稀释的一抗， 孵育60min 或过夜(湿盒内); (2) 酶标抗体间接法的操作步骤(续) *0.01M PBST漂洗，5min×3; *加HRP标记的二抗室温孵育lh或; *加0.01,H2O2~0.05,DAB显色 (显色液应新鲜配置); *经PBS漂洗3次后，梯度酒精脱水，二甲苯透明，明胶甘油封片，显微镜观察。 (2) 非标记抗体酶法——酶桥法 *首先用酶免疫动物，制备效价高、特异性强的抗酶抗体; *以二抗作桥，将抗酶抗体联结在一抗上; *再将酶结合在抗酶抗体上，经显色显示抗原的分布 -优点:任何抗体均未被酶标记。酶是通过免疫学原理与酶抗体结合的。避免了共价连接对抗体和酶活性的损害，提高了方法的敏感性，而且节省一抗的用量。但抗酶抗体不易纯化。 几点说明 *一抗(假设来自种属A)的稀释度可大些，使抗体的两个Fab段均与组织抗原结合。 *二抗——桥抗体(抗种属A的IgG抗体)应过量，使其Fab段一个与一抗结合，另一个则游离。 *因抗酶抗体与一抗均系种属A IgG，具有相同的抗原性，所以桥抗体游离的Fab能与抗酶抗体结合，起桥作用，将其连接在与组织抗原结合的一抗上。 (2) 非标记抗体酶法——PAP法 * 与酶桥法相似，不同的是，PAP法将酶桥法的第3、4步并为1步，用PAP复合物代替; *PAP是离体制备的复合物(HRP--抗HRP)。 *显色与酶桥法相同，PAP复合物中的过氧化物酶催化底物水解，形成不溶性终产物。 PAP法评价 *PAP法比直接法、间接法、酶桥法更敏感。特别适用于石蜡切片中微量抗原和抗原性减弱抗原的检测。 -缺点:步骤较多，时间较长，不适用于临床常规检查。 *由于常做石蜡切片，故可用于回顾性研究。 (三) 亲和组织化学法 *是以一种物质对某种组织成分具有高度亲合力为基础。 *这种方法敏感性更高，有利于微量抗原(抗体)在细胞或亚细胞水平的定位。 生物素—抗生物素染色法 【原理】 *生物素(biotin)又称维生素H，是一种小分子维生素，分子量为244，是转氨甲酰基化过程 中的辅酶。 *抗生物素(avidin)，又称卵白素或亲和素，是一种分子量为67000的碱性蛋白，对生物素具有很强的亲和力，比抗原抗体间的亲和力要高出100万倍。它由4个亚基组成，每个亚基都有生物素的结合位点。 *两者均可与抗体等大分子生物活性物质相偶联，又可被酶类等多种示踪物所标记，形成生物素-抗生物素系统。 -该系统一端偶联大分子生物反应体系，另一端连结标记物，后者加入酶的底物，产生颜色反应。 (1)标记抗生物素—生物素法(labelled avidin-biotin method, LA:分为直接法和间接法。 *直接法 用生物素标记第一抗体，与抗原结合;酶标记抗生物素，与生物素结合，然后进行酶呈色反应。 * 间接法 用生物素标记二抗，酶标记抗生物素，先用第一抗体与组织抗原结合，再将第二抗体与第一抗体相连结，最后进行呈色反应。 (2)桥抗生物素一生物素法(bridge avidin-biotin method，BRA -此法是用生物素分别标记抗体和酶，以抗生物素为桥，把二者连接起来，进行呈色反应。 (3) 抗生物素-生物素-过氧化物酶法 (ABC法) *ABC法是在BRAB和LAB的基础上改良的方法。 *ABC复合物是将过氧化物酶结合在生物素上，再将其与过量的抗生物素反应而制备的。 *分为直接法和间接法。 -直接法是生物素标记的一抗与ABC复合物结合; -间接法是生物素标记的二抗与ABC复合物结合。 ABC法的评价 *敏感性强:ABC法比PAP法敏感性高20~40倍。 *特异性强，背景染色淡:由于敏感性高，一抗和二抗都可被稀释至可能的浓度，减少了非特异染色。 *方法简便，节约时间。可由PAP法所需的2天缩短至几个小时。 *由于生物素与抗生物素具有与多种示踪物结合的能力，可用于双重或多重免疫。 试验方法 本文共包括三部分:1、酶免疫组化实验;2、荧光免疫组化实验;3、实验问题分析 PART ONE Enzymatic Protocol 1 Contents , Overview , Materials , Sample Preparation , Tissue Fixation and Mounting - Cryostat Sections , Tissue Fixation and Mounting - Paraffin-embedded Sections , Tissue Staining Protocol - Chromogenic , Support Protocols , Gel Coating Solution , Gel-coated Slides , Antigen-retrieval Protocol Overview R&D Systems provides monoclonal, polyclonal and biotinylated antibodies for immunohistochemical use. The following protocol has been developed and optimized by R&D Systems' Immunohistochemical Laboratory. R&D Systems' antigen affinity-purified polyclonal and monoclonal antibodies have been used to stain frozen cells and tissues, as well as paraffin-embedded tissues. Immunohistochemistry protocols may require modification depending on the type of tissue used. Each investigator should determine the optimal conditions and working dilutions of antibodies. If using R&D Systems' primary antibodies, refer to the specific product insert to obtain an approximate working dilution. For all other reagents, follow the manufacturer's instructions. For Research Use Only. Materials Primary Antibodies Unlabeled or biotinylated antigen affinity-purified polyclonal antibodies (R&D Systems' AF or BAF series) or selected unlabeled or biotinylated monoclonal antibodies (R&D Systems' MAB or BAM series) Cell & Tissue Staining Kits - Enzymatic/Chromogenic , Against Goat Primary Antibodies: , AP-BCIP/NBT (R&D Systems Catalog # CTS007) , HRP-DAB (R&D Systems Catalog # CTS008) , HRP-AEC (R&D Systems Catalog # CTS009) , Against Mouse Primary Antibodies: , AP-BCIP/NBT (R&D Systems Catalog # CTS001) , HRP-DAB (R&D Systems Catalog # CTS002) , HRP-AEC (R&D Systems Catalog # CTS003) , Against Rabbit Primary Antibodies: , AP-BCIP/NBT (R&D Systems Catalog # CTS004) , HRP-DAB (R&D Systems Catalog # CTS005) , HRP-AEC (R&D Systems Catalog # CTS006 Buffers and Additional Supplies , Fixative: 85 mM Na2HPO4, 75 mM KH2P04, 4% formaldehyde (Sigma Catalog # P6148) and 14% (v/v) saturated picric acid (Sigma Catalog # 925-40), pH 6.9. Picric acid is optional. , Sucrose Solution: 130 mM Na2HPO4, 30 mM KH2PO4, 10% (w/v) sucrose (Sigma Catalog # S7903), 0.01% sodium azide (Sigma Catalog # S2002) and 0.03% Bacitracin (Sigma Catalog # B-0125), pH 7.2 , PBS: 50 mM Na2HPO4 and 140 mM NaCl, pH 7.2 , Incubation Buffer: 1% bovine serum albumin (Sigma Catalog # A2153), 1% normal donkey serum (Sigma Catalog # D9663), 0.3% Triton X-100 (Sigma Catalog # T9284) and 0.01% sodium azide (Sigma Catalog # S2002) in PBS , Aqueous Mounting Medium (R&D Systems Catalog # CTS011) , DAB Enhancer (R&D Systems Catalog # CTS010) , Antigen Retrieval Reagents (R&D Systems Catalog # CTS013, CTS014, CTS015 or CTS016) Note: Equivalent chemicals and reagents may be substituted for those listed above. Sample Preparation (Frozen Tissues) The vast majority of immunohistochemical procedures employ a cell or tissue fixation step using formaldehyde or other cross-linking fixatives prior to incubation with primary antibody. Fixation is required to retain tissue morphology and prevent degradation of tissue antigens. Fixation may be performed either by immersing dissected pieces of tissue (e.g. human biopsies) into the fixative, or by vascular perfusion (e.g. laboratory animals such as mice, rats, guinea pigs, etc.). It is very important to optimize fixing conditions since under- or over-fixation may reduce or abolish tissue immunoreactivity. The easiest way to correct under-fixation is to post-fix tissue sections on the slide before starting immunohistochemical staining. To recover antigens in over-fixed tissues, either protease-induced epitope retrieval (PIER) or heat-induced epitope retrieval (HIER) techniques are recommended. HIER can be performed using a microwave oven, pressure cooker, vegetable steamer, autoclave or water bath. After tissues are fixed, they may either be embedded into paraffin or covered with OCT compound and frozen for further sectioning. Paraffin-embedded tissues are cut using a microtome at room temperature, whereas frozen tissues are cut using a cryostat at temperatures below 0?C. Antigen immunoreactivity was found to be better preserved in frozen rather than 1,2paraffin-embedded tissues. References 1. Larsson, L.-I. (1988) Immunocytochemistry: Theory and Practice, CRC Press, Boca Raton, Florida. 2. Frost, A. et al. (2000) Methods of antigen recovery vary in their usefulness in unmasking specific antigens in immunohistochemistry, Appl. Immunohistochem. Mol. Morphol. 8:236. Tissue Fixation and Mounting - Cryostat Sections 1. Fix the tissue by vascular perfusion with 500 - 700 mL of Fixative. 2. Perfuse the animal with 400 mL of Sucrose Solution. 3. Dissect the tissue, mount in OCT and freeze at -20 to -80?C. 4. Cut 5 - 15 mm thick tissue sections using a cryostat. 5. Thaw-mount the sections onto gel-coated slides. Refer to the Support Protocols section to follow for instructions on how to prepare gel-coated slides. 6. Dry the slides for 30 minutes on a slide warmer at 37?C. Slides containing cryostat sections can be stored at -20 to -70?C for up to 12 months. Tissue Fixation and Mounting - Paraffin-embedded Sections 1. Fix the tissue by vascular perfusion with 500 - 700 mL of Fixative. 2. Dissect the tissue. 3. Immerse the tissue in 70% ethanol three times for 30 minutes each at room temperature. 4. Immerse the tissue in 90% ethanol two times for 30 minutes each at room temperature. 5. Immerse the tissue in 100% ethanol three times for 30 minutes each at room temperature. 6. Immerse the tissue in toluene three times for 20 minutes each at room temperature. 7. Embed the tissue in paraffin (Paraplast, Fisher Scientific) two times for 60 minutes each at 58?C. Alternatively, tissues can be embedded into paraffin using specialized automated tissue processing systems. 8. Cut 5 - 15 祄 thick tissue sections using a rotary microtome. 9. Float the sections in a 56?C water bath. Mount the sections onto histological slides. 10. 11. Dry the slides overnight at room temperature. Slides containing paraffin-embedded sections can be stored at room temperature. When it is not possible to fix tissue by perfusion, dissected tissue may be fixed by immersing the tissue into a 10% formalin solution for 4 - 8 hours at room temperature. It is commonly accepted that the volume of fixative should be 50 times greater than the size of the immersed tissue. Avoid fixing the tissue for greater than 24 hours since tissue antigens may either be destroyed or masked (A.C. Cuello, ed., 1993, Immunohistochemistry: Methods in the Neurosciences, Vol. 14; IBRO Handbook Series, John Wiley & Sons, New York). Tissue Staining Protocol - Chromogenic This protocol is based on using R&D Systems' AP-BCIP/NBT Cell & Tissue Staining Kits. Refer to the protocol booklet provided with each kit for technical notes. Steps may vary depending on the chromogenic substrate. Refer to the Materials section of this protocol for additional substrates. 1. Prepare the slides as follows. 1. Cryostat tissue sections Thaw the slides for 10 minutes at room temperature. 2. Paraffin-embedded tissue sections 1. Immerse the slides in Xylene (mixed isomers) two times for 10 minutes each. 2. Immerse the slides in 100% alcohol (denatured) two times for 10 minutes each. 3. Immerse the slides in 95% alcohol once for 5 minutes. 4. Immerse the slides in 70% alcohol once for 5 minutes. 5. Immerse the slides in 50% alcohol once for 5 minutes. 6. Rinse the slides in deionized water. 2. Rehydrate the slides in PBS for 10 minutes. Drain the slides and wipe off excess buffer. 3. 4. Perform antigen-retrieval, if necessary. Refer to the Support Protocols section to follow. 5. Incubate the sample with 1 - 3 drops of Serum Blocking Reagent for 15 minutes. 6. Drain the slides and wipe off excess reagent. Do not rinse. 7. Incubate the sample with 1 - 3 drops of Avidin Blocking Reagent for 15 minutes. 8. Rinse the sample with PBS. Drain the slides and wipe off excess buffer. 9. Incubate the sample with 1 - 3 drops of Biotin Blocking Reagent for 15 minutes. 10. Rinse the sample with PBS. Drain the slides and wipe off excess buffer. 11. Incubate the sample with primary antibody diluted in Incubation Buffer. Follow the manufacturer's recommended working dilution, incubation time and incubation temperature. Note: An overnight incubation at 4?C is recommended when using primary antibodies from R&D Systems. 12. Rinse the sample with PBS and then wash 3 times in PBS for 5 minutes each. Drain the slides and wipe off excess buffer. 13. Incubate the sample with 1 - 3 drops of Secondary Biotinylated Antibodies for 30 - 60 minutes. Adjust the incubation time depending on the thickness of the tissue section. 14. Rinse the sample with PBS and then wash 3 times in PBS for 15 minutes each. Drain the slides and wipe off excess buffer. 15. Incubate the sample with 1 - 3 drops of HSS-AP for 30 minutes. 16. Rinse the sample with PBS and then wash 3 times in PBS for 2 minutes each. 17. Rinse the sample with distilled water. Drain the slides and wipe off excess water. 18. Incubate the sample with 1 - 5 drops of BCIP/NBT Chromogen for 20 - 30 minutes. Monitor the intensity of tissue staining under a microscope. 19. Rinse the sample with PBS and then wash in PBS for 10 minutes. 20. Rinse the sample with distilled water. Drain the slides and wipe off excess water. 21. Mount the sample either without counterstain or after counterstaining with Methyl Green or Nuclear Fast Red. 22. Drain the excess mounting medium by placing the slides vertically on filter paper or a paper towel. Allow the slide to dry. 23. Examine the slides under a microscope. Support Protocols Preparation of gel-coated slides Gel Coating Solution: 1. Dissolve 5 g gel [type A, 175 bloom from porcine (Sigma Catalog # G2625)] in 1 L water, while heating. Do not allow the temperature to exceed 45?C. 2. Add 0.5 g chromium potassium sulfate [CrK(SO4)2 . 12H2O] and dissolve completely. Store at 4?C. This solution can be re-used 3 - 4 times and is stable for 1 - 2 months when stored at 4?C. Gel-coated Slides: 1. Heat the Gel Coating Solution to 40 - 44?C. 2. Filter the solution using coarse filter paper. 3. Pour the solution into a large staining dish. 4. Remove bubbles from the surface of the solution. 5. Briefly submerge a rack containing histological slides into the solution. 6. Remove the rack of slides from the staining dish. 7. Place the rack onto a paper towel and cover the slides with paper towels to avoid contamination with dust. 8. Allow the slides to dry at room temperature. Dry slides can be stored for 1 year at -20?C. Antigen-retrieval Protocol This protocol is based on using R&D Systems' Antigen Retrieval Reagents (Catalog # CTS013, CTS014, CTS015 or CTS016). Note: the antigen-retrieval capacity of each Antigen Retrieval Reagent depends on sample preparation, antigen structure, incubation time (up to 30 minutes) and temperature (90 - 100?C). Each investigator should determine optimal conditions. 1. Dilute the 10X Antigen Retrieval Reagent 10-fold, using deionized water, to make the Retrieval Solution. 2. Heat the Retrieval Solution to 92 - 95?C. This may be accomplished by placing a polypropylene Coplin staining jar filled with Retrieval Solution into a water bath. Note: heating may crack glass staining dishes. 3. Insert the slides containing tissues into the heated Retrieval Solution and incubate 2 - 10 minutes. Note: cryostat sections are more susceptible to the damaging effects of the Retrieval Solution than paraffin-embedded tissues. To avoid tissue damage, it may be necessary to shorten the incubation time to 2 - 5 minutes for cryostat sections.4. Place the Coplin jar containing the Retrieval Solution and slides on a lab bench and allow to cool for 5 - 10 minutes at room temperature. 5. Rinse the slides with distilled water followed by PBS. Note: tissues may become loose after the retrieval procedure, avoid vigorous rinsing to prevent detachment of the tissues from the slides. Proceed to step 5 of the Tissue Staining Protocol. Enzymatic Protocol 2 Contents , Overview , Materials , Sample Preparation , Tissue Staining Overview R&D Systems provides monoclonal, polyclonal and biotinylated antibodies for immunohistochemical applications. Antigen affinity-purified polyclonal antibodies are recommended for immunohistochemistry because they are 10-fold more potent than the traditional protein G affinity-purified polyclonal antibodies. The use of analyte-specific affinity chromotography greatly reduces or totally eliminates non-specific immunoglobulins. The benefits of using antigen-affinity purified polyclonal antibodies are increased staining intensity and reduced non-specific staining. The following protocol has been developed and optimized by R&D Systems?Immunohistochemical Laboratory. R&D Systems?antigen affinity-purified polyclonal and monoclonal antibodies have been used on frozen rat brain tissues as well as on paraffin-embedded human tissues. This protocol may need to be modified depending on the type of tissue used. Individual investigator should determine optimal working dilution of antibodies. If using R&D Systems?primary antibodies, refer to product specification sheets to obtain approximate working dilutions. For all other reagents, follow manufacturer抯 instructions. For Research Use Only. Materials Primary Antibodies , Unlabeled or biotinylated antigen affinity-purified polyclonal antibodies (R&D Systems?AF or BAF series) or selected monoclonal antibodies Secondary Antibodies , Biotinylated donkey anti-goat IgG (Cat# 705-065-147; Jackson ImmunoResearch Laboratories) Buffers and Additional Supplies , Fixative: 4% formaldehyde (Catalog # P6148; Sigma) and of 14% (v/v) saturated picric acid in 0.16 M phosphate buffered saline (PBS), pH 6.9 , Sucrose Solution: 10% sucrose solution in 0.1 M PBS (pH 7.2) , Incubation Buffer: 0.1 M PBS, pH 7.4 containing 1% bovine serum albumin, 1% normal donkey serum, 0.3% Triton X-100 and 0.01% sodium azide , Wash Buffer: 0.1 M PBS, pH 7.4 , Avidin-Biotin Blocking Kit: (SP-2001, Vector Labs) , Vectastain Elite ABC-Peroxidase Kit: (Cat# PK-6105; Vector Labs) , Substrates: AEC (Cat# SK-4200; Vector Labs), DAB (SK-4100), VIP (SK-4600), NovaRED (SK-4800, Vector Labs) , Mounting Media: Use an aqueous or a non-aqueous mounting media of choice Sample Preparation (Frozen Tissues) 1. Fix tissue by vascular perfusion with 500 - 700 mL Fixation Buffer. 2. Perfuse tissue with 400 mL Sucrose Solution. 3. Cut tissue using a cryostat at a nominal thickness of 5??5牭m and thaw-mount onto histological slides. 4. Dry slides with tissue sections for 30 minutes on a slide warmer at 37?C. 5. Store slides in a freezer at -20 to -70?C. Tissue Staining: Chromogenic 1. Thaw slides at room temperature for 10 minutes. 2. Rehydrate the slides with Wash Buffer for 10 minutes. 3. Drain excess Wash Buffer. 4. Tissue suspected of binding avidin and biotin should be treated with Avidin-Biotin Blocking Kit, according to manufacturer's instructions. 5. Apply primary antibodies diluted in Incubation Buffer and incubate at 4癈 for 24 - 72 hours. Note: A negative control and/or an isotype matched control should be performed, which will identify non-specific binding from the secondary antibody. A negative control is the incubation buffer with no primary antibody. An isotype control may be employed when using monoclonal antibodies. 6. Wash slides 3 times for fifteen minutes each in Wash Buffer. Proceed to step 9 if a biotinylated antibody was used in step 5. 7. Incubate with the secondary antibody for 30 - 60 minutes at room temperature. Apply biotinylated secondary antibody if an unlabeled primary antibody was used. 8. Wash slides 3 times for fifteen minutes each in Wash Buffer. 9. Apply Vectastain ABC Elite Kit according to manufacturer's instructions. 10. Rinse slides once in Wash Buffer. 11. Prepare and apply Substrate of choice followed by a counterstaining of nuclei. 12. Wash tissue according to manufacturer's instructions specified for the Substrate. 13. Use an aqueous or a non-aqueous mounting media suitable for the Substrate. Note: Alcohol washes out certain substrates, therefore, follow manufacturer's specifications for Substrate to prevent wash out. Enzymatic Protocol 3 Contents , Materials , Controls , Staining Tissue , Sample Preparation and Fixation , Antibody Incubations , Staining Cells Smeared on Slides , Sample Preparation and Fixation , Antibody Incubations , Immunoenzymatic Technique , Frequently Asked Questions , Technical Hints , References Materials Cytokine-specific Primary Antibodies , unlabeled or biotinylated antigen-affinity purified polyclonal antibodies R&D Systems 'AF or BAF series or selected monoclonal antibodies. Secondary Antibodies and Secondary Reagents , Biotinylated donkey anti-goat IgG (Jackson Immuno Research Lab Catalog # 705-066-147) , Biotinylated goat anti-mouse IgG Caltag Lab Catalog # M32115) 1 , Vectastain Elite-ABC-peroxidase (Vector Lab Catalog # PK6100) , ExtrAvidin-HRP (Sigma Catalog # E2886) Substrate , 3,3'-Diaminobenzidine tetra hydrochloride (DAB) (Sigma Catalog # D5637) Buffers and Additional Supplies , Fixation Buffer Formaldehyde (37% v/v Sigma) is diluted in PBS to a final formaldehyde concentration of 2% (v/v) and adjust pH to 7.4. Light sensitive, store at 4癈 in the dark; prepare working dilution just prior to fixation , Wash Buffer Earls Buffered Salt Solution (EBSS) (Gibco BRL) , Wash Buffer-Saponin EBSS with 0.1% (w/v) Saponin (Sigma Catalog # S4521) and adjust pH to 7.2-7.4 with NaOH. , Endogenous Peroxidase Blocking Buffer 3M NaN in EBSS with 1% HOand 322 0.1% (w/v) Saponin. , Endogenous Biotin Blocking Buffer Avidin/Biotin Blocking Kit (Vector Lab Catalog # SP2001). Both Avidin D and Biotin should be supplemented with 0.1% (w/v) Saponin. , Immunohistochemistry Mounting Medium PBS buffered glycerol 1:9 (v/v) and adjust pH to 7.4. , Mayer's Hematoxylin Counterstain (Sigma Catalog # MHS-16) , Saponin stock Prepare a stock of 10 % (w/v) Saponin in EBSS. Prepare fresh, since crude saponin powder batches are generally fungi infected. , Microscope slides Adhesion slides (BioRad Catalog # 12550-15) (smeared cells); TC microscope glass slides (tissue sections); HTC slides (Cel-line Assoc. Catalog # 10-618) , Frozen tissue embedding OCT-compound (VWR Catalog # 25608-930) , Humidified Chamber A plastic light protected box lined with wet paper towels. Controls Positive staining controls , Stain fixed, permeabilized cytokine-cDNA transfected eukaryotic cells expressing the intra-cellular target cytokine protein. , Stain cultured blood mononuclear cells or spleen cells that have been harvested, fixed and permeabilized at the peak of cytokine production after in vitro stimulation with strong +polyclonal activators such as phorbol 12-myristate 13 acetate/ionomycin, anti-CD3CD28 monoclonal antibody, LPS or bacterial superantigens such as staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPE-A). Negative staining controls , Ligand blocking control: to study the specificity of the cytokine staining by a preincubation of the cytokine-specific antibody with its target cytokine at a molar ratio of 1:10 prior to addition to the cells. , Isotype matched control immunoglobulin: Stain the cells by a primary isotype pre-matched immunoglobulin of irrelevant antigen-specificity at the same concentration as the cytokine-specific antibodies. , Unlabeled antibody control: Cells that will be stained by a fluorochrome or a biotinylated primary cytokine-specific antibody will be incubated initially with the unconjugated version of the same antibody. , Non transfected eukaryotic cells and unstimulated PBMNC are good negative controls. Cytokine Detection in Tissue using Immunohistochemistry This method is based on cryopreserved tissue, which after sectioning will be fixed in formaldehyde and stained by cytokine-specific antibodies by indirect immunoenzymatically based technique. It has proven mandatory to include saponin in all antibody incubations, despite the fact that the tissue has been cryostat-sectioned. Sample preparation and fixation 1. Immediately snap freeze fresh tissue in isopentane in dry ice and kept at -70癈. Do not allow frozen tissue to thaw before cutting. 2. Embed tissue completely in OCT compound prior to cryostat sectioning. 3. Cut cryostat sections at 5-10 祄 and mount on HTC glass slides.Note: Suggested cryostat temperature is -23癈 and the tissue specimen is -18癈. The section will curl if the specimen is too cold and if it is too warm it will stick to the knife. 4. Air dry the sections for 30 minutes at room temperature to prevent sections from falling off the slides during antibody incubations.Note: Store the slides unfixed for several months at -70癈. Frozen tissue samples for analysis at later time points should be stored unsectioned. 5. Immediately add 50 礚 of ice-cold Fixation buffer on each tissue section upon removal from the freezer. 6. Fix for 8 minutes at 4癈 or less optimally at 20癈 for 20 minutes. Antibody Incubations 1. Wash samples three times with Wash Buffer-Saponin for three minutes each. 2. Block endogenous peroxidase activity in the specimens with Endogenous Peroxidase Blocking Buffer for 30-60 minutes at room temperature in the dark. 3. Wash three times with Wash Buffer-Saponin for three minutes each. 4. Block endogenous biotin with Endogenous Biotin Blocking Buffer for 30 minutes in a sequential mode. Prepare both the Avidin D and the Biotin by adding 6 drops/mL of the stock solution to Wash Buffer-Saponin. Note: Incubation with avidin D may need to be prolonged in tissue with high endogenous biotin activity. The diluted biotin should be supplemented with 2% serum from the same species as the source of the secondary antibody. 5. Wash sample twice with Wash Buffer-Saponin. 6. Incubate overnight (at least 12 hours) with 50-100 礚 of primary antibody (0.5-5.0 礸 /mL in Wash Buffer-Saponin) or negative control antibody in a humidified chamber at room temperature. It is essential that the specimens will not dry out. 7. Wash samples three times with Wash Buffer-Saponin for three minutes each. 8. Incubate for 30-45 minutes at room temperature with 25-50 礚 of a biotinylated secondary antibody (either biotin-donkey anti-goat IgG diluted 1:700 or biotin-goat anti-mouse IgG or IgG or IgGdiluted 1:500) in Wash Buffer-Saponin. 12A2B 9. Wash slides three times in Wash-Buffer-Saponin for 30 seconds each. 10. Prepare Vectastain Elite ABC-peroxidase reagent according to manufacturers' instructions supplemented with saponin (0.1% w/v) 30 minutes prior to incubation. 11. Incubate with Vectastain Elite ABC-peroxidase for 30 minutes at room temperature in the dark. 12. Wash samples three times with Wash Buffer without saponin for three minutes prior to substrate incubation. 13. Prepare the substrate according to manufacturers instructions without any supplementation of saponin. 14. Incubate sample for approximately 8 minutes (monitor in the microscope). A brown color reaction with distinct morphology is developed with DAB in the peroxidase system. 15. Stop the development of the color reaction by repeated washes in distilled water. 16. Counterstain, if desired, with hematoxylin for 1-5 seconds. Slides are then air dried and can be mounted and coverslipped with Immunohistochemistry Mounting Medium without any further dehydration. Slides that need to be kept for long periods in optimal shape can be dehydrated in ethanol and mounted in special mounting medium. Immunostaining of individual cytokine-producing cells smeared on slides Suitable for use on single-cell suspensions from peripheral blood, lymphoid tissue or cultured cell-lines. Sample Preparation and Fixation 1. Harvest cells and wash twice in V-bottomed tubes with cold Wash Buffer by centrifugation (400 x g for 5 minutes) to remove extracellular proteins, including cytokines. 62. Resuspend to 1-5 x 10 cells/mL in Wash Buffer. Transfer 10-15 礚 of the cell suspension to each reaction field on the adhesion slide. 3. 4. Allow the cells to adhere electrostatically in a monolayer for 10 minutes at room temperature in the humidified chamber to prevent the cells from drying out. 5. Add 50 礚 of ice-cold Fixation Buffer to each field to fix the cells. 6. Incubate for 20 minutes at 4癈. 7. Wash three times with Wash Buffer to remove free formaldehyde. 8. Add 25 礚 of 2% fetal bovine serum in Wash Buffer to block unbound surface area on the slide. 9. Incubate for 10 minutes at 37癈. 1. Wash slides three times in Wash Buffer-Saponin. The slides are now ready for staining. 2. Alternatively, wash the slides in Wash Buffer and allow the slides to dry. Dried slides can be stored at -20癈 several months before being stained. Prior to staining the slides should be washed in Wash Buffer-Saponin. Antibody Incubation All antibody incubations and washes are performed in Wash Buffer-Saponin to keep the cells permeable for antibodies to penetrate the cell membranes. Detection using Biotin-labeled antibodies 1. Incubate in Endogenous Peroxidase Blocking Buffer for 30 minutes at room temperature in the dark to block endogenous peroxidase activity in the cells (this step can be omitted if cells are to be stained by fluorochromes or non-peroxidase based enzymatic methods). 2. Block endogenous biotin activity with the Avidin/Biotin blocking kit in a two step procedure for 30爉inutes in the presence of saponin, described in steps 3-5. 3. Incubate each cell spot on slides with Avidin for 15 minutes supplemented with saponin (0.1% w/v). 4. Wash each cell spot on slides twice in Wash Buffer-Saponin. 5. Incubate in Biotin for 15 minutes supplemented with saponin (0.1% w/v). 6. Wash each cell spot on slides twice in Wash Buffer-Saponin. 7. Incubate each cell spot on slides for 30 minutes at room temperature with 15 礚 unlabeled or biotinylated cytokine-specific antibodies (0.5-5 礸/mL) diluted in Wash Buffer-Saponin. 8. Wash slides three times in Wash Buffer-Saponin. Note: If using R&D Systems biotinylated antibody skip steps 9 and 10 and continue. Incubate each cell spot on slides for 30 minutes at room temperature with 15 礚 of a 9. biotinylated secondary antibody (either biotin-donkey anti-goat IgG Fab diluted 1:700; or 2 biotin-goat anti mouse IgG or IgG or IgG diluted 1:500) in Wash Buffer-Saponin. 12A2B 10. Wash slides three times in Wash Buffer-Saponin. Cytokine-specific staining based on either biotinylated primary antibodies or unlabeled primary antibodies along with biotinylated secondary antibodies can then be developed by techniques based on immunoflourescence or immunoenzymatic methods. Immunoenzymatic Technique 1. Prepare Vectastain Elite ABC-peroxidase reagent according to manufacturers' instructions and supplement with saponin (0.1% w/v), 30 minutes prior to step 2. 2. Incubate for 30 minutes with Vectastain Elite ABC-peroxidase at room temperature in the dark. 3. Wash slides three times with distilled water prior to substrate incubation. 4. Prepare the substrate according to the the protocol given by the manufacturer without any supplementation of saponin. 5. Incubate for approximately 8 minutes (monitor in the microscope) at room temperature in the dark. A brown color reaction with distinct morphology is developed with DAB in the peroxidase system. 6. Stop the development of the color reaction by repeated washes in distilled water. 7. Counterstain, if desired, with hematoxylin for 1-5 seconds. Slides are then air dried and coverslipped with Immunohistochemistry Mounting Medium. Slides that need to be kept for long periods in optimal shape should be dehydrated in ethanol and mounted in special mounting medium. Frequently Asked Questions Question 1 Question: Are background signals caused by the developing substrate? Test: Incubate with substrate alone. Cause of Background: Endogenous enzyme (peroxidase/alkaline phosphatase) activity in sample Remedy: Block with Endogenous Peroxidase Blocking Buffer (with Levamisol in the alkaline phosphate system). Question 2 Question: Are background signals caused by endogenous biotin activity in the sample? Test: (Block if necessary as in Question 1) Incubate with Vectastain ABC (or ExtraAvidin HRP/AP) and incubate with substrate. Cause of background: Endogenous biotin activity. Remedy: Block with Endogenous Biotin/Avidin Blocking-Buffer. Question 3 Question: Does the secondary biotinylated antibody cause background signals? Test: (Block if necessary as in Question 1 and/or Question 2). Incubate with the following three steps: secondary antibody; Vectastain ABC-kit (or ExtrAvidin HRP/AP); and substrate. Cause of background: Fc-receptor binding of secondary antibody used at too high concentration. Remedy: Block with species matched serum to secondary antibody or try to dilute the secondary antibody. Question 4 Question: Does the primary cytokine specific antibody generate background signals? Test: (Block if necessary as in Question 1 and/or Question 2). Use secondary antibody at optimal concentration. Incubate with the following: primary antibody; secondary antibody; Vectastain ABC (or ExtrAvidin HRP/AP); and with substrate. Cause of background: Cytokine-detection antibody used at too high concentration or unsuitable for immunostaining. Remedy: Titrate antibody or try another cytokine detecting antibody. Question 5 Question: Is the cytokine staining specific? Test: Incubate cytokine-detecting antibody with target cytokine overnight and finally add 0.1 (saponin prior to staining as in Question 4. Cause of Background: Cytokine-detecting antibody used at too high concentration or unsuitable for immunostaining. Remedy: Titrate antibody or try another cytokine detecting antibody. Technical Hints Permeabilization: It is crucial that saponin is present during all antibody incubations and washes to make the staining procedure successful. For detection of intracellular cytokines, the cytokine-specific antibodies must penetrate through the cell surface membrane, the cytosol, the membranes of the endoplasmic reticulum and the Golgi organelle. The detergent, saponin has been shown to intercalate in the membranes to replace cholesterol and to permeabilize cells in a reversible way, maintaining much of the morphology of the membrane structure of the cell. Fixation: A solution of phosphate-buffered formaldehyde has been found to preserve cell morphology as well as surface and intracellular antigenicity with minuscule cell aggregation and cell loss. Only fixed cells will stand the effects of detergent treatment. Controls: Evidence for specificity of the cytokine staining should be based on parallel studies of isotype controls, staining with the secondary antibodies alone and an abolishment of immunoreactivity by preabsorption of the cytokine-specific antibody with the corresponding cytokine protein. Stimulation of PBMNC for cytokine production: The strongest and most diversified cytokine production is seen after PMA-ionomycin activation of Ficoll separated PBMNC. Both monocytes and lymphocytes will be activated. The cells are co-cultivated with PMA (1 ng/mL), ionomycin (500 nM/mL) and harvested after 4 hours. After 4 hours, one can expect to see IL-1a, IL-1b, IL-1ra, IL-2, IL-3, for various periods. If cells are harvested after 3 hours, one can find monokines such as IL-1a, IL-1b, IL-1ra, IL-6, IL-8, MIP-1a, MIP-1b, and TNF-a. However, cytokines produced by lymphocytes, IL-12 or substantial IL-10 production is not found in the cultures. References 1. Litton, M. et al. (1997) Am. J. Pathol. 150(5):1607. 2. Litton, M. et al. (1997) Human Cytokine Protocols, Debets/Savelkoul eds., Humana Press, USA, In press. 3. Sparrelid, E. et al. (1997) Transplantation 63:1. 4. Lundberg, I. et al. (1997) Arthritis Rheum 40(5):865. 5. Bj鰎k, L. et al. (1996) J. Leukocyte Biol. 59(2):287. 6. Litton, M. et al. (1996) Eur. J. Immunol 25(1):1. 7. 舓erlund, K. et al. (1996) Scand. J. Immunol. 44:345. 8. Behringer, D. et al. (1996) Histochem. J. 28:461. 9. Raqib, R. et al. (1995) Infection Immunity 63:289. 10. Ulfgren, A-K. et al. (1995) Ann. Rheum. Dis. 54:654. 11. Andersson, U. et al. (1994) Cytokine Producing Cells, eds., D. Fradelizi et al., INSERM, France., p. 32. 12. Fernandez, V. et al. (1994) Eur. J Immunol. 24:1808. 13. Andersson, J. et al. (1994) Immunology 82:16. 14. Dolhain, R.J.E.M. et al. (1993) J. Leukocyte Biol. 54:545. 15. Sander, B. et al. (1993) J. Immunol. Methods 166:201. 16. Andersson, J. et al. (1992) Immunol. Rev. 127:69. 17. Abrams, J. et al. (1992) Immunol. Rev. 127:5. 18. Andersson, J. et al. (1992) Eur. Immunol. 22:2617. Sander, B. et al. (1991) Immunol. Rev. 119:65. 19. 20. Andersson, U. et al. (1990) Eur. J. Immunol. 20:1591. 21. Fischer, H. et al. (1990) J. Immunol. 144:4663. 22. Sander, B. et al. (1989) Scand. J. Immunol. 30:315. 23. Henter, J. et al. (1988) Eur. J. Immunol. 18:983. 24. Andersson, U. et al. (1988) Eur. J. Immunol. 18:2081. PART TWO Fluorescence Protocol 1 Contents , Overview , Materials , Sample Preparation , Tissue Staining Overview R&D Systems provides monoclonal, polyclonal and biotinylated antibodies for immunohistochemical use. Antigen affinity-purified polyclonal antibodies are recommended for immunohistochemistry because they are 10-fold more potent than the traditional protein G affinity-purified polyclonal antibodies. The use of analyte-specific affinity chromotography greatly reduces or totally eliminates non-specific immunoglobulins. The benefits of using antigen-affinity purified polyclonal antibodies are increased staining intensity and reduced non-specific staining. The following protocol has been tested by R&D Systems?Immunohistochemistry Laboratory. R&D Systems?antigen affinity-purified polyclonal and monoclonal antibodies have been used on frozen rat brain tissues as well as on paraffin-embedded human tissues.The protocol may need to be modified depending on the type of tissue used. Individual investigator should determine optimal working dilution of antibodies. If using R&D Systems?primary antibodies, refer to product specification sheets to obtain approximate working dilutions. For all other reagents, follow manufacturer抯 instructions. Due to accumulation of autofluorescent pigment lipofuscin in primate and human neuronal tissues, the use of fluorescent probes, for example, FITC or Cy3 is not recommended. Protocols using chromogens (e.g., DAB, AEC or immunogold-silver staining) may be used instead. For Research Use Only. Materials Primary antibodies , Unlabeled or biotinylated antigen-affinity purified polyclonal antibodies R&D Systems?AF or BAF series or selected monoclonal antibodies Secondary antibodies , Donkey anti-goat Cy3-conjugated antibodies (Cat# 705-165-147; Jackson ImmunoResearch Laboratories) or equivalent , Donkey anti-chicken Cy3-conjugated antibodies (Cat# 703-165-155; Jackson ImmunoResearch Laboratories) or equivalent , Donkey anti-mouse FITC conjugated antibodies (Cat# 715-095-151; Jackson ImmunoResearch Laboratories) or equivalent , Donkey anti-goat FITC conjugated antibodies (Cat# 705-095-147; Jackson ImmunoResearch Laboratories) or equivalent , Streptavidin conjugated to Cy3 (Cat# 016-160-084; Jackson ImmunoResearch Laboratories) or equivalent , Streptavidin conjugated to FITC (Cat# 016-090-084; Jackson ImmunoResearch Laboratories) or equivalent Buffers and additional supplies , Fixation Buffer: 4% formaldehyde (P6148; Sigma) and of 14% (v/v) saturated picric acid in 0.16 M phosphate buffered saline (PBS), pH 6.9 , Sucrose Solution: 10% sucrose solution in 0.1 M PBS (pH 7.2) , Incubation Buffer: 0.1 M PBS, pH 7.4 containing 1% bovine serum albumin, 1% normal donkey serum, 0.3% Triton X-100 and 0.01% sodium azide , Avidin-Biotin Blocking Kit: (SP2001, Vector Labs) , Wash Buffer: 0.1 M PBS, pH 7.4 , Mounting Media: an anti-fade mounting media of choice Sample Preparation 1. Fix tissue by vascular perfusion with 500 - 700 mL Fixation Buffer. 2. Perfuse tissue with 400 mL Sucrose Solution. 3. Cut tissue using a cryostat at a nominal thickness of 5 - 15 祄 and thaw-mount onto histological slides. 4. Dry slides with tissue sections for 30 minutes on a slide warmer at 37?C. 5. Store slides in a freezer at -20 - -70?C. Tissue Staining 1. Thaw slides at room temperature for 10 minutes. 2. Rehydrate the slides with Wash Buffer for 10 minutes. 3. Drain excess Wash Buffer. 4. Tissue suspected of binding nonspecifically avidin should be treated with Avidin-Biotin Blocking Kit, according to manufacturer抯 instructions. Do not employ avidin blocking step when using biotinylated primary antibodies. 5. Apply primary antibodies diluted in Incubation Buffer and incubate at 4癈 for 24 ?72 hours. Note: A negative control and/or an isotype matched control should be performed, which will identify non-specific binding from the secondary antibody. A negative control is the incubation buffer with no primary antibody. An isotype control may be employed when using monoclonal antibodies. 6. Wash slides 3 times for fifteen minutes each in Wash Buffer. 7. Incubate with the secondary antibody. Apply Streptavidin conjugated to Cy3 if a biotinylated antibody was used in step 5. 8. Wash slides 3 times for fifteen minutes each in Wash Buffer. 9. Mount with an anti-fade mounting media. Fluorescence Protocol 2 Contents , Materials , Controls , Staining Cells in Suspension , Sample Preparation and Fixation , Antibody Incubations , Staining Cells Smeared on Slides , Sample Preparation and Fixation , Antibody Incubations , Immunofluorescent Technique , Frequently Asked Questions , Technical Hints , References Materials Cytokine-specific Primary Antibodies , unlabeled or biotinylated antigen-affinity purified polyclonal antibodies (R&D Systems 'AF' or 'BAF' series) or selected monoclonal antibodies. Secondary Antibodies and Secondary Reagents , Biotinylated donkey anti-goat IgG (Jackson Immuno Research Lab Catalog # 705-066-147) , Biotinylated goat anti-mouse IgG (Caltag Lab Catalog # M32115) 1 , FITC-labeled goat anti-mouse IgG(Caltag Lab Catalog # M32001) 1 , FITC-labeled anti-mouse IgG (Caltag Lab Catalog # M32201) 2A , FITC-labeled anti-mouse IgG (Caltag Lab Catalog # M32401) 2B , Oregon Green coupled anti-mouse IgG (Molecular Probes Catalog # 06380) , Oregon Green-avidin D(Molecular Probes Catalog # A6374) , FITC-Avidin D (Sigma Catalog # A-2001) Buffers and Additional Supplies , Fixation Buffer Formaldehyde (37% v/v Sigma) is diluted in PBS to a final formaldehyde concentration of 2% (v/v) and adjust pH to 7.4. Light sensitive, store at 4癈 in the dark; prepare working dilution just prior to fixation , Wash Buffer Earls Buffered Salt Solution (EBSS) (Gibco BRL) , Wash Buffer-Saponin EBSS with 0.1% (w/v) Saponin (Sigma Catalog # S4521) and adjust pH to 7.2-7.4 with NaOH. , Endogenous Peroxidase Blocking Buffer 3M NaN in EBSS with 1% HOand 322 0.1% (w/v) Saponin. , Endogenous Biotin Blocking Buffer Avidin/Biotin Blocking Kit (Vector Lab Catalog # SP2001). Both Avidin D and Biotin should be supplemented with 0.1% (w/v) Saponin. , Fluorescence Anti Fading Mounting Medium Carbonate/bicarbonate buffered glycerol (1:1 v/v) containing 2 % 1,4-Diazobicyclo 2.2 octane (Sigma Catalog # D2522) and adjust pH to 7.4. , Saponin stock Prepare a stock of 10 % (w/v) Saponin in EBSS. Prepare fresh, since crude saponin powder batches are generally fungi infected. , Intracellular transport inhibitor Brefeldin A (Sigma Catalog # B7651) , Microscope slides Adhesion slides (BioRad Catalog # 12550-15) (smeared cells); TC microscope glass slides (tissue sections); HTC slides (Cel-line Assoc. Catalog # 10-618) , Frozen tissue embedding OCT-compound (VWR Catalog # 25608-930) , Humidified Chamber A plastic light protected box lined with wet paper towels. Contents Controls Positive staining controls , Stain fixed, permeabilized cytokine-cDNA transfected eukaryotic cells expressing the intra-cellular target cytokine protein. , Stain cultured blood mononuclear cells or spleen cells that have been harvested, fixed and permeabilized at the peak of cytokine production after in vitro stimulation with strong +CD28 polyclonal activators such as phorbol 12-myristate 13 acetate/ionomycin, anti-CD3 monoclonal antibody, LPS or bacterial superantigens such as staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPE-A). Negative staining controls , Ligand blocking control: to study the specificity of the cytokine staining by a preincubation of the cytokine-specific antibody with its target cytokine at a molar ratio of 1:10 prior to addition to the cells. , Isotype matched control immunoglobulin: Stain the cells by a primary isotype pre-matched immunoglobulin of irrelevant antigen-specificity at the same concentration as the cytokine-specific antibodies. , Unlabeled antibody control: Cells that will be stained by a fluorochrome or a biotinylated primary cytokine-specific antibody will be incubated initially with the unconjugated version of the same antibody. , Non transfected eukaryotic cells and unstimulated PBMNC are good negative controls. Contents Immunofluorescent staining of cytokine-producing cells in suspension Cells intended for analysis by flow cytometry should be cultured in vitro prior to harvest in the presence of an intracellular transport inhibitor such as Brefeldin A (1-5 礸/mL). This step will increase the concentration of intracellular cytokines, which will enhance the discrimination of cytokine-positive cells from background signals caused by autofluorescence. The treatment will, however, ruin the characteristic morphology, seen in the UV-microscope, of a rounded perinuclear dot generated by the the accumulation of the cytokines in the Golgi organelle. Computerized image analysis offers an alternative to score these cells by automated technique without any need for cell cultures with Brefeldin A. The computer program is designed in such a way that the system will record single cells as positive or negative cytokine producers based on the intensity and the color of the cytokine staining combined with an appreciation of the characteristic morphology of the juxtanuclear staining pattern generated by the accumulation of the cytokines in the Golgi organelle. Sample Preparation and Fixation 1. Harvest cells and wash twice in V-bottomed tubes with cold Wash Buffer by centrifugation (400 x g for 5 minutes) at 4癈 to remove extracellular cytokines. 2. Add 0.5 mL of the Fixation Buffer to the cell pellet and incubate for 20 minutes. 3. Note: During the fixation time, the cells are vortexed to avoid cell aggregation. 4. Wash the cells twice in Wash Buffer Saponin by centrifugation (400 x g for 5 minutes). 6For cytokine staining aliquot cells at a concentration of 1-5 x 10/mL in tubes or 5. microplates. Fixed cells can also be frozen in culture medium supplemented with 15% fetal bovine serum and 10% dimethylsulfoxide (DMSO) for future cytokine staining. Antibody Incubation All antibody incubations and washes should be performed in Wash Buffer-Saponin to keep the cells permeable for antibodies to penetrate the cell membranes. a. Detection using Biotin-labeled antibodies 1. Block endogenous biotin activity with the Avidin/Biotin blocking kit in a two step procedure for 30 minutes in the presence of saponin, described in steps 2-4 below. 2. Incubate cells with Avidin for 15 minutes supplemented with saponin (0.1% w/v). 3. Wash the cells twice in Wash Buffer-Saponin by centrifugation (400 x g for 5 minutes). 4. Incubate the cells in Biotin for 15 minutes supplemented with saponin (0.1% w/v). 5. Wash the cells twice in Wash Buffer-Saponin by centrifugation (400 x g for 5 minutes). 6. Add 50 礚 of unlabeled or biotinylated cytokine-specific antibodies (0.5-5 礸/mL) diluted in Wash Buffer-Saponin to the vortexed cell pellet. 7. Incubate for 30 minutes at room temperature. 8. Wash twice in Wash Buffer-Saponin by centrifugation (400 x g for 5 minutes). 9. Incubate the cells for 30 minutes at room temperature with 50 礚 of a biotinylated secondary antibody (either biotin-donkey anti-goat IgG Fab diluted 1:700 or biotin-goat 2 anti-mouse IgG or IgG or IgG diluted 1:500) in Wash Buffer-Saponin. 12A2B 10. Wash the cells twice in Wash Buffer-Saponin. 11. Incubate the centrifuged cell pellet for 30 minutes at room temperature with 50 礚 of Oregon-Green-Avidin D or FITC-Avidin D (2-5 礸/mL Wash Buffer-Saponin). 12. Wash twice in Wash Buffer-Saponin by centrifugation (400 x g for 5 minutes) followed by a final wash in Wash Buffer by centrifugation without any saponin (400 x g for 5 minutes). Cells are then ready for analysis by flow cytometry or alternatively by UV-microscopy. 13. For UV microscopy analysis the cells can be resuspended in 0.1 M sodium citrate and drops of the suspension can be put on glass slides and be left to air-dry. The dried cell preparations can be mounted in Anti-Fading Buffer to reduce UV quenching of FITC. Oregon-Green does not fade and can stand daylight without problem. In addition it gives a very crisp and intensive signal. Stained cells can be stored at 4癈 for several weeks without loss of staining quality. It is possible to perform multiple color-staining in the same cells using different fluorochromes by using this staining protocol sequentially. Between staining for different antigens, block with the Biotin/Avidin kit to prevent binding to biotinylated antibodies from the previous staining. b. Detection using fluorochrome-labeled antibodies If cytokine producing cells are to be detected by fluorochrome-labeled primary or secondary antibodies there is no need to block endogenous biotin activity. Background signals are often reduced when fluorochrome-labeled antibodies are used in Wash Buffer-Saponin supplemented with 5% human AB serum. 1. Incubate the suspended cells for 30 minutes at room temperature with 50 礚 of unlabeled or fluorochome-labeled primary cytokine-specific antibodies (0.5-5 礸/mL) diluted in Wash Buffer-Saponin supplemented with 5% human AB serum. 2. Wash the cells twice by centrifugation in Wash Buffer-Saponin (400 x g for 5 minutes). 3. Incubate cells for 30 minutes at room temperature with 15 礚 of a fluorochrome-labeled secondary antibody (either FITC-labeled anti-mouse IgG, IgG or 12A IgG diluted 1:300) in Wash Buffer-Saponin supplemented with 5% human AB serum. 2B 4. Cells are ready for analysis by flow cytometry or alternatively by UV-microscopy. Contents Immunostaining of individual cytokine-producing cells smeared on slides Suitable for use on single-cell suspensions from peripheral blood, lymphoid tissue or cultured cell-lines. Sample Preparation and Fixation 1. Harvest cells and wash twice in V-bottomed tubes with cold Wash Buffer by centrifugation (400 x g for 5 minutes) to remove extracellular proteins, including cytokines. 62. Resuspend to 1-5 x 10 cells/mL in Wash Buffer. 3. Transfer 10-15 礚 of the cell suspension to each reaction field on the adhesion slide. 4. Allow the cells to adhere electrostatically in a monolayer for 10 minutes at room temperature in the humidified chamber to prevent the cells from drying out. 5. Add 50 礚 of ice-cold Fixation Buffer to each field to fix the cells. 6. Incubate for 20 minutes at 4癈. 7. Wash three times with Wash Buffer to remove free formaldehyde. 8. Add 25 礚 of 2% fetal bovine serum in Wash Buffer to block unbound surface area on the slide. Incubate for 10 minutes at 37癈. 9. 1. Wash slides three times in Wash Buffer-Saponin. The slides are now ready for staining. 2. Alternatively, wash the slides in Wash Buffer and allow the slides to dry. Dried slides can be stored at -20癈 several months before being stained. Prior to staining the slides should be washed in Wash Buffer-Saponin. Antibody Incubation All antibody incubations and washes are performed in Wash Buffer-Saponin to keep the cells permeable for antibodies to penetrate the cell membranes. a. Detection using Biotin-labeled antibodies 1. Incubate in Endogenous Peroxidase Blocking Buffer for 30 minutes at room temperature in the dark to block endogenous peroxidase activity in the cells (this step can be omitted if cells are to be stained by fluorochromes or non-peroxidase based enzymatic methods). 2. Block endogenous biotin activity with the Avidin/Biotin blocking kit in a two step procedure for 30爉inutes in the presence of saponin, described in steps 3-5. 3. Incubate each cell spot on slides with Avidin for 15 minutes supplemented with saponin (0.1% w/v). 4. Wash each cell spot on slides twice in Wash Buffer-Saponin. 5. Incubate in Biotin for 15 minutes supplemented with saponin (0.1% w/v). 6. Wash each cell spot on slides twice in Wash Buffer-Saponin. 7. Incubate each cell spot on slides for 30 minutes at room temperature with 15 礚 unlabeled or biotinylated cytokine-specific antibodies (0.5-5 礸/mL) diluted in Wash Buffer-Saponin. 8. Wash slides three times in Wash Buffer-Saponin. Note: If using R&D Systems biotinylated antibody skip steps 9 and 10 and continue. 9. Incubate each cell spot on slides for 30 minutes at room temperature with 15 礚 of a biotinylated secondary antibody (either biotin-donkey anti-goat IgG Fab diluted 1:700; or 2 biotin-goat anti mouse IgG or IgG or IgG diluted 1:500) in Wash Buffer-Saponin. 12A2B 10. Wash slides three times in Wash Buffer-Saponin. Cytokine-specific staining based on either biotinylated primary antibodies or unlabeled primary antibodies along with biotinylated secondary antibodies can then be developed by techniques based on immunoflourescence or immunoenzymatic methods. Immunofluorescent Technique 1. Incubate slides for 30 minutes at room temperature with 15 礚 of Oregon-Green Avidin or FITC-Avidin at a concentration of 2-5 礸/mL in Wash Buffer-Saponin. 2. Wash slides twice in Wash Buffer-Saponin. 3. Wash slides once in Wash Buffer only. Allow slides to air dry before mounting in Mounting Buffer. FITC staining, but not Oregon-Green staining, will require a mounting medium including some anti-fading substance to reduce UV quenching. It is possible to store fluorochrome-stained slides for long periods in the freezer, provided that they have not been mounted in Mounting medium. b. Detection using fluorochrome-labeled antibodies If cytokine producing cells are to be detected by fluorochrome-labeled primary or secondary antibodies there is no need to block endogenous peroxidase or biotin activity. Background signals are often reduced when fluorochrome-labeled antibodies are used in Wash Buffer-Saponin supplemented with 5% human AB serum. 1. Incubate each cell spot on slides for 30 minutes at room temperature with 15 礚 of either unlabeled or fluorochome-labeled primary cytokine-specific antibodies (0.5-5 礸/mL) diluted in Wash Buffer-Saponin supplemented with 5% human AB serum. 2. Wash slides three times in Wash Buffer-Saponin. 3. Incubate cells for 30 minutes at room temperature with 15 礚 of flurochrome-labeled secondary antibody (either FITC-labeled anti-mouse IgG or IgG or IgG diluted 1:300) in 12A2B Wash Buffer-Saponin supplemented with 5% human AB serum. 4. Wash slides three times in Wash Buffer-Saponin and air dry the slides. 5. Mount and coverslip with Fluorescence Anti Fading Mounting Medium. Contents Frequently Asked Questions Question 1 Question: Is the cytokine staining specific? Test: Incubate cytokine-detecting antibody with target cytokine overnight and finally add 0.1 (saponin prior to staining as in Question 4. Cause of Background: Cytokine-detecting antibody used at too high concentration or unsuitable for immunostaining. Remedy: Titrate antibody or try another cytokine detecting antibody. Contents Technical Hints Permeabilization: It is crucial that saponin is present during all antibody incubations and washes to make the staining procedure successful. For detection of intracellular cytokines, the cytokine-specific antibodies must penetrate through the cell surface membrane, the cytosol, the membranes of the endoplasmic reticulum and the Golgi organelle. The detergent, saponin has been shown to intercalate in the membranes to replace cholesterol and to permeabilize cells in a reversible way, maintaining much of the morphology of the membrane structure of the cell. Fixation: A solution of phosphate-buffered formaldehyde has been found to preserve cell morphology as well as surface and intracellular antigenicity with minuscule cell aggregation and cell loss. Only fixed cells will stand the effects of detergent treatment. Controls: Evidence for specificity of the cytokine staining should be based on parallel studies of isotype controls, staining with the secondary antibodies alone and an abolishment of immunoreactivity by preabsorption of the cytokine-specific antibody with the corresponding cytokine protein. Stimulation of PBMNC for cytokine production: The strongest and most diversified cytokine production is seen after PMA-ionomycin activation of Ficoll separated PBMNC. Both monocytes and lymphocytes will be activated. The cells are co-cultivated with PMA (1 ng/mL), ionomycin (500 nM/mL) and harvested after 4 hours. After 4 hours, one can expect to see IL-1a, IL-1b, IL-1ra, IL-2, IL-3, for various periods. If cells are harvested after 3 hours, one can find monokines such as IL-1a, IL-1b, IL-1ra, IL-6, IL-8, MIP-1a, MIP-1b, and TNF-a. However, cytokines produced by lymphocytes, IL-12 or substantial IL-10 production is not found in the cultures. Contents References 1. Litton, M. et al. (1997) Am. J. Pathol. 150(5):1607. 2. Litton, M. et al. (1997) Human Cytokine Protocols, Debets/Savelkoul eds., Humana Press, USA, In press. 3. Sparrelid, E. et al. (1997) Transplantation 63:1. 4. Lundberg, I. et al. (1997) Arthritis Rheum 40(5):865. 5. Bj鰎k, L. et al. (1996) J. Leukocyte Biol. 59(2):287. 6. Litton, M. et al. (1996) Eur. J. Immunol 25(1):1. 7. 舓erlund, K. et al. (1996) Scand. J. Immunol. 44:345. Behringer, D. et al. (1996) Histochem. J. 28:461. 8. 9. Raqib, R. et al. (1995) Infection Immunity 63:289. 10. Ulfgren, A-K. et al. (1995) Ann. Rheum. Dis. 54:654. 11. Andersson, U. et al. (1994) Cytokine Producing Cells, eds., D. Fradelizi et al., INSERM, France., p. 32. 12. Fernandez, V. et al. (1994) Eur. J Immunol. 24:1808. 13. Andersson, J. et al. (1994) Immunology 82:16. 14. Dolhain, R.J.E.M. et al. (1993) J. Leukocyte Biol. 54:545. 15. Sander, B. et al. (1993) J. Immunol. Methods 166:201. 16. Andersson, J. et al. (1992) Immunol. Rev. 127:69. 17. Abrams, J. et al. (1992) Immunol. Rev. 127:5. 18. Andersson, J. et al. (1992) Eur. Immunol. 22:2617. 19. Sander, B. et al. (1991) Immunol. Rev. 119:65. 20. Andersson, U. et al. (1990) Eur. J. Immunol. 20:1591. 21. Fischer, H. et al. (1990) J. Immunol. 144:4663. 22. Sander, B. et al. (1989) Scand. J. Immunol. 30:315. 23. Henter, J. et al. (1988) Eur. J. Immunol. 18:983. 24. Andersson, U. et al. (1988) Eur. J. Immunol. 18:2081. PART THREE Troubleshooting Guide: Problem: Lack of Staining Check protein expression by in situhybridization Lack of antigen (in some rare cases translation may be blocked even though mRNA is detected) Aliquot antibodies into smaller volumes sufficient Antibodies do not work due to to make a working solution for a single improper storage experiment. Store aliquots in a freezer (-20 to -70 癈) and avoid repeated freeze-thaw cycles Inactive primary antibodies Replace with a new batch of antibodies Inadequate tissue fixation Try different fixative Tissue overfixation Reduce duration of postfixation or fix tissues at 4?C. If postfixation can not be avoided (for example, collection of postmortem tissues in pathology lab) antigens may be unmasked by treatment with antigen-retrieval reagents Incompatible secondary and Use secondary antibody that will interact with the primary antibodies primary antibody. For example, if primary antibodies were raised in rabbits, use anti-rabbit secondary but not anti-mouse or anti-goat Inactive secondary reagents Replace with new reagents Antigen was destroyed before Quenching of endogenous peroxidase was done incubation with primary antibody prior to the addition of primary antibodies. Block peroxidase after incubation with primary antibody Problem: Overstaining High concentration of primary Determine optimal concentration for each and/or secondary antibodies component of immunohistochemical reaction: and/or reagent primary antibodies; secondary antibodies; enzymes catalyzing formation of color precipitates Long incubation time Determine optimal incubation time for each component of immunohistochemical reaction: primary antibodies; secondary antibodies; enzymes; chromogenic substrates Non-specific binding of primary Treat tissues to reduce or block non-specific and/or secondary reagents to binding of immunohistochemical components to tissues tissues, for example, by Avidin-Biotin Blocking Reagents Problem: High Background Tissues have endogenous Incubate with normal serum obtained from molecules which are also used species other than from the species that were the in incubation mixtures, for source of the primary antibody example, the presence of peroxidase in blood cells or the Block endogenous component. Endogenous presence of autofluorescent peroxidase may be blocked by incubating tissues pigment lipofuscin in neuronal for 30 minutes at room temperature with 0.3% cells H2O2 in methanol, or switch to protocols utilizing substances which are not present in tissues For autofluorescence: after finishing IHC staining, treat tissues with 1% Sudan Black in 70% alcohol. If residual autofluorescence still obscures specific labeling, use non-fluorescent enzymatic protocols with chromogenic substrates (for instance DAB or AEC) or employ immunogold-silver staining 免疫组化的试剂准备 admin 最重要的是选择好一抗、二抗或者还有三抗，注意抗体的特异性、效价和交叉反应，最好用 单抗。 其他常用试剂有: 1、0.01M(pH7.4)的磷酸盐缓冲生理盐水(PBS)，稀释抗体和洗片子用; 2、清洁液、纯水，洗玻片用 3、多聚赖氨酸处理载玻片，防止脱片 4、固定液:4%多聚甲醛或冷丙酮等; 5、15%~30%蔗糖，组织脱水用; 6、梯度酒精脱水、二甲苯透明、石蜡包埋，或者OCT包埋做冰冻切片; 7、抗原修复液:枸橼酸缓冲液(pH6.0); 8、活化剂:EDTA或者Triton X-100; 9、1%~10%牛血清清蛋白(BSA)封闭液; 10、HO，灭活内源性过氧化物酶; 22 11、显色液:根据你做的酶来选择，如果是HRP就用DAB，如果是AKP，就用NBT/BCIP， 免疫荧光则不需要; 12、50%缓冲甘油封片液。 免疫细胞化学常用试剂介绍 佚名 一、固定剂 大多数神经激素、肽类物质为水溶性，在用于免疫细胞化学研究之前，常需固定。但肽类和蛋白质的物理、化学性质不同，因而对不同的固定方法或固定剂的反应也不尽相同。某些固定剂甚至可同时破坏和/或保护同一抗原的不同抗原决定簇。因此，在进行免疫细胞化学研究之前，很有必要了解所要研究的物质(蛋白质或肽类)的化学性质，并根据需要来选择适宜的固定剂(或固定方法)以及改进固定条件。 目前，免疫细胞化学研究中常用的固定剂仍为醛类固定剂，其中以甲醛类和戊二醛最为常用。在此，简要介绍几种目前较为常用和推荐的固定剂，以供读者选用。 1.4%多聚甲醛-0.1mol/L磷酸缓冲液(pH7.3) 试剂:多聚甲醛 40g 0.1mol/L磷酸缓冲液 至1000ml 配制方法:称取40g多聚甲醛，置于三角烧瓶中，加入500,800ml 0.1mol/L磷酸缓冲液(Phosphate Buffer以下简称PB)，加热至60?左右，持续搅拌(或磁力搅拌)使粉末完全溶解，通常需滴加少许1n NaOH才能使溶液清亮，最后补足0.1mol/L的PB于1000ml，充分混匀。 该固定剂较适于光镜免疫细胞化学研究，最好是动物经灌注固定取材后，继续浸泡固定2,24h。另外，该固定剂较为温和，适于组织标本的较长期保存。 2.4%多聚甲醛-磷酸二氢钠/氢氧化钠 试剂:A液:多聚甲醛 40g 蒸馏水 400ml B液:Na2HPO4?2H2O 16.88g 蒸馏水 300ml C液:NaOH 3.86g 蒸馏水 200m 配制方法:A液最好在500ml的三角烧瓶中配制(方法同前)，至多聚甲醛完全溶解后冷却待用。注意，在溶解多聚甲醛时，要尽量避免吸入气体或溅入眼内。B液和C液配制 好后，将B液倒入C液中，混合后再加入A液，以1n NaOH或1N HCl 将pH调至7.2,7.4，最后，补充蒸馏水至1000ml充分混合，4?冰箱保存备用。 该固定剂适于光镜和电镜免疫细胞化学研究，用于免疫电镜时，最好加入少量新鲜配制的戊二醛，使其终浓度为0.5%,1%。该固定剂较温和，适于组织的长期保存。组织标本于该固定液中，4?冰箱保存数月仍可获得满意的染色效果。 3(Bouin’s液及改良Bouin’s液 试剂:饱和苦味酸 40%甲醛 250ml 冰醋酸 50ml 配制方法:先将饱苦味酸过滤，加入甲醛(有沉淀者禁用)，最后加入冰醋酸，混合后存于4?冰箱中备用。冰醋酸最好在临用前加入。改良Bouin’s液即不加冰醋酸。 该固定液为组织学、病理学常用固定剂这一，对组织的穿透力较强，固定较好，结构完整。但因偏酸(pH为3,3.5)，对抗原有一定损害，且组织收缩较明显，故不适于组织标本的长期保存。此外，操作时，应避免吸入或与皮肤接触。 4(Zamboni’s(Stefanini’s)液 试剂:多聚甲醛 20g 饱和苦味酸 150ml Karasson-Schwlt’s PB 至1000ml 配制方法:称取多聚甲醛20g，加入饱和苦味酸150ml，加热至60?左右，持续搅拌使充分溶解、过滤、冷却后，加Karasson-Schwlt’s PB至1000ml充分混合(Karasson –Schwlt’s磷酸缓冲液的配制配制方法见后)。 该固定液适于电镜免疫细胞化学，对超微结构的保存较纯甲醛为优，也适于光镜免疫细胞化学研究，为实验室常用固定剂之一。我们在应用中，常采用2.5%的多聚甲醛和30%的饱和苦味酸，以增加其对组织的穿透力和固定效果，保存更多的组织抗原。固定时间为6,18h。 5(PLP液 PLP液即过碘酸盐-赖氨酸-多聚甲醛固定液 (Periodate-Lysine-paraform-alde,hyde fixative) 试剂:过碘酸钠 赖氨酸盐酸盐(或盐酸赖氨酸): 多聚甲醛 蒸馏水 配制方法: (1)贮存液A(0.1mol/L赖氨酸-0.5mol/l Na3PO4,pH7.4):称取赖氨酸盐酸盐1.827g溶于50ml蒸馏水中，得0.2mol/L的赖氨酸盐酸盐溶液，然后加入Na2HPO4至0.1mol/L，将pH调至7.4，补足0.1mol/L的PB至100ml，使赖氨酸浓度也为0.1mol/L,4?冰箱保存，最好两周内使用。此溶液的渗透浓度为300mOs mmol/L/kg。 (2)贮存液B(8%多聚甲醛溶液):称8g多聚甲醛加入100ml蒸馏水中，配成8%多聚甲醛液(方法见前)。过滤后4?冰箱保存。 (3)临用前，以3份A液与1份B液混合，再加入结晶过碘酸钠(NaIO4)，使NaIO4终浓度为2%。由于AB两液混合，pH从约7.5降至6.2，故固定时不需再调pH值。固定时间为6,18h。 该固定剂较适于富含糖类的组织，对超微结构及许多抗原的抗原性保存较好。其机制是借助于过碘酸氧化组织中的糖类形成醛基，通过赖氨酸的双价氨基与醛基结合，从而与糖形成交联。由于组织抗原绝大多数都是由蛋白质和糖两部分构成，抗原决定簇位于蛋白部分，故该固定剂有选择性地使糖类固定，这样既稳定了抗原，又不影响其在组织中的位置关系。Mclean和Nakane等认为，最佳的混合是:含0.01mol/L的过碘酸盐、0.075mol/L的赖氨酸、2%的多聚甲醛及0.037mol/L的磷酸缓冲液。 6(Karnovsky’s液(pH7.3) 试剂:多聚甲醛 30g 25%戊二醛 80ml 0.1mol/L PB 至1000ml 配制方法:先将多聚甲醛溶于0.1mol/L PB中，再加入戊二醛，最后加0.1mol/L的PB至1000ml，混匀。 该固定剂适于电镜免疫细胞化学，用该固定液在4?短时固定，比在较低浓度的戊二醛中长时间固定能更好地保存组织的抗原性和细微结构。固定时最好先灌注固定，接着浸泡固定10,30min，用缓冲液漂洗后即可树酯包埋或经蔗糖溶液后用于恒冷切片。 7(0.4% 对苯醌 (Parabenzoquinone) 试剂:对苯醌 4.0g 0(01mol/L PBS 1 000ml 配制方法:称取4.0g对苯溶于1000ml 0.01mol/L的PBS即可。 对苯醌对抗原具有较好的保护作用，但对超微结构的保存有一定影响，故常与醛类固定剂混合使用。一般要求临用前配制，且避免加热助溶，因加热或放置时间过长，固定液变为棕色至褐色，会使组织标本背景增加，影响观察。此外，对苯醌有剧毒，使用时避免吸入或与皮肤接触。 8(PFG液(Parabenzoquinone-Formaldehyde-Glutaraldehyde fixative, PFG) 试剂:对苯醌 20g 多聚甲醛 15g 25%戊二醛 40ml 0.1mol/L二甲酸钠缓冲液 至1000ml 配制方法:先以500ml左右的二甲酸钠缓冲液溶解对苯醌及多聚甲醛，然后加入戊二醛，最后加入二甲酸钠缓冲液至1000ml充分混合。 对苯醌与戊二醛及甲醛联合应用，即可阻止醛基对抗原的损害，又不影响超微结构的保存，故适于多种类抗原的免疫细胞化学，尤其是免疫电镜的研究。 9(碳二亚酰胺-戊二醛(ECD-G)液 试剂:0.05mol/L PB 500ml 0.01mol/L PBS 500ml Tris 约14g 浓HCl 少许 ECD 10g 25%戊二醛 3.5ml 配制方法:先以约500ml的PB与相同体积的PBS混合，加入Tris(使其终浓度为1.4%)溶解，以浓HCl调pH至7.0，再将事先称取好的ECD和戊二醛加入混合液，振摇后计时，用pH计检测，约2,3min时，pH降至6.6，再以1N的NaOH在4min内调pH至7.0，此时，将该混合固定液加入盛有细胞(经PBS漂洗过)的器皿中，在23?固定7min后，以PBS洗去固定液，即可进一步处理。 ECD即乙基-二甲基氨基丙基碳亚胺盐酸盐[1-ethyl-3(3-dimethyl-aminoprpyl)-carboi,imide hydrochloride]，简称乙基-CDI，常用于多肽类激素的固定，对酶等蛋白质的固定也有良好效果。ECD单独应用时，边缘固定效应重，但与戊二醛、Tris及PB联合应用，效果明 显改善，细胞质仍可渗透，利用细胞中抗原的定位，超微结构保存较好。目前认为是一种用于培养细胞电镜水平免疫细胞化学研究的很好的固定剂。 10(四氧化锇(锇酸Osmic Acid, OsO4) 配制:将洗净装有OsO4的安瓿中加热后，迅速投入装有溶剂的棕色瓶中，使安瓿遇冷自破。也可用钻石刀在安瓿上划痕，洗净后再放入瓶中，盖好瓶塞，用力撞击安瓿，待其破后加溶剂稀释。为保证充分溶解，应在用前几天配制。 (1)2% OsO4水溶解:取OsO41g溶于重蒸水中。此液常作为储备液，于冰箱中密封保存。 (2)1% OsO4-PB: 试剂:A、2.26% NaH2PO4?2H2O 4.15ml B2.52% 8.5ml C、5.4% 葡萄糖 5ml D、OsO4 0.5g 配制方法:先分别配好A、B、C三种液体，取A液41.5ml与B液8.5ml混合，将pH调至7.3,7.4，取A,B混合液45ml再与5ml C液混合即为0.12mol/L PBG。 (3)1% OsO4/0.1mol/L二甲胂酸钠缓冲液(pH7.2,7.4): 试剂:2% OsO4水溶液 10ml 0.2mol/L二甲胂酸钠缓冲液(pH7.2,7.4) 10ml 配制方法:取2%OsO4储备液10ml与等量0.2mol/L、pH7.2,7.4的二甲胂酸钠缓冲液充分混合即可。 OsO4是电镜研究所必需的试剂，常用于后固定。尽管OsO4主要为脂类固定剂，但也可与肽类及蛋白质起作用，形成肽-蛋白质或肽-脂交联。过氧化物酶的反应产物经OsO4处理后，电子密度增高，适于电镜研究。但由于OsO4的反应产物对光及电子有较明显的吸收能力，因此在免疫细胞化学染色前常需去除，去锇在光镜水平常用1%的高锰酸钾，在电镜水平则常用H2O2来处理。 以上介绍了目前免疫细胞化学中常用的一些固定液。关于固定液种类还很多，如70%,90%的酒精、丙酮、醋酸酒精(含0.1%,1%醋酸的70%,90%的酒精等)，这些溶液都能促使蛋白质凝固。它们最初只是光学显微镜通用的固定液，但在免疫细胞化学上用其它方法不成功时，也可试用。总之，掌握一个原则，免疫细胞化学中，含重金属的固定液禁用(但Zenker-Formalin可进行短时的固定)。目前多数认为，对生物标本较好的固定措施是:用4? 的Karnovsky’s液灌注固定10,30min后，接着在pH7.3、0.1mol/L的二甲胂酸钠缓冲液中漂洗过液，这种短时冷固定处理，有助于超微结构和许多肽类抗原的保存。对其它较难保存的抗原可尝试PFG、PLP及Zamboni’s液等混合固定液。 二、缓冲液 免疫细胞化学中应用的缓冲液种类较多，即使是同种缓冲液，其浓度、pH、离子强度等也常常不同。在此介绍几种最常用的缓冲液的配制。 1(0.2mol/L(pH7.4)磷酸盐缓冲液(Phosphate Buffer, PB) 试剂: NaH2PO4?2H2O Na2HPO4?12H2O 配制方法:配制时，常先配制0.2mol/L的 NaH2PO4和0.2mol/L的Na2HPO4，两者按一定比例混合即成0.2mol/L的磷酸盐缓冲液(PB)，根据需要可配制不同浓度的PB和PBS。 (1)0.2mol/L的 Na2HPO4;称取Na2HPO4.12H2o 31.2g(或 NaH2PO4?H2O 27.6g)加重蒸水至1000ml溶解。 (2)0.2mol/L的 Na2HPO4:称取NaHPO4.?12H2o 71.632g(或 Na2HPO4?7H2O 53.6g 或 Na2HPO4?2H2o 35.6g)加重蒸水至1000ml溶解。 (3)0.2mol/L pH7.4的PB的配制:取19ml 0.2mol/L的 NaH2PO4和81ml 0.2mol/L的 Na2HPO4?12H2O,充分混合即为0.2mol/L的PB(pH约为7.4,7.5)。若pH偏高或偏低，可通过改变二者的比例来加以调整，室温保存即可。 2(0.01mol/L磷酸盐缓冲生理盐水(Phosphate Buffered Saline, PBS) 试剂:0.2mol/L PB 50ml NaCl 8.5,9g(约0.15mol/L) 重蒸水 至1000ml 配制方法:称取NaCl 8.5,9g及0.2mol/L的PB 50ml，加入1000ml的容量瓶中，最后加重蒸水至1000ml，充分摇匀即可。若拟配制0.02mol/L的PBS，则PB量加倍即可，依此类推。 PB和PBS是免疫细胞化学实验中最为常用的缓冲液，0.01mol/L的PBS主要用于漂洗组织标本、稀释血清等，其pH应在7.25,7.35之间，否则需要调整。0.1mol/L的PB常用于配制固定液、蔗糖等。一般情况下，0.2mol/l PB的pH值稍高些，稀释成0.01mol/L的PBS时，常可达到要求的pH值，若需调整pH，通常也是调PB的pH。 3(Karasson –Schwlt’s磷酸盐缓冲液 试剂: NaH2PO4?H2O 3.31g Na2HPO4?7H2O 33.77g 重蒸水 至1000ml 配制方法:同前 该缓冲液主要用于配制Zamboni’s固定液。 4(0.5mol/L pH7.6的Tris- HCl缓冲液。 试剂:Tris(三羟甲基氨基甲烷) 60.57g 1N HCl 约420ml 重蒸水 至1000ml 配制方法:先以少量重蒸水(300,500ml)溶解Tris,加入HCl后，用1N的HCl或1N的NaOH将pH调至7.6，最后加重蒸水至1000ml。此液为储备液，于4?冰箱中保存。免疫细胞化学中常用的Tris-HCl缓冲液浓度为0.05mol/L，用时取储备液稀释10倍即可。 该液主要用于配制Tris缓冲生理盐水(TBS)、DAB显色液。 5(Tris缓冲生理盐水(Tris Buffered,Saline,TBS) 试剂:0.5mol/L Tris-HCl缓冲液 100ml NaCl 3.5,9g(0.15mol/L) 重蒸水 至1000ml 配制:先以重蒸水少许溶解NaCl,再加Tris-HCl缓冲液，最后加重蒸水至1000ml，充分摇匀使Tris终浓度为0.05mol/L。 TBS主要用于漂洗标本，常用于免疫酶技术中。 6(Tris-TBS(PBS) 试剂:Triton X-100 10ml(1%)或3ml(0.3%) 0.5mol/L Tris缓冲液(pH7.6) 1000ml(50ml)或(0.2mol/L的PB) NaCl 8.5,9g 重蒸水 至1000ml 配制方法:先以重蒸水少许溶解NaCl后，加入Triton X-100及Tris缓冲液或(PB)，最后加重蒸水至1000ml，充分摇匀。 该液常用浓度为1%及0.3%，前者主要用于漂洗标本，后者主要用于稀释血清。 7(0.1mol/L(pH7.4)二甲胂酸缓冲液 试剂:0.2mol/L二甲胂酸钠 500ml 0.1N HCl 28ml 蒸馏水 至1000ml 配制方法:先称取二甲胂酸钠(MW:214)42.8g,加蒸馏水至1000ml，使0.2mol/L的二甲胂酸钠溶液;再取HCl 1.7ml加蒸馏水至1000ml ,配成0.1N，最后 取0.2mol/L二甲胂酸钠溶液500ml及0.1n HCl 28ml混合，加蒸馏水至1000ml，即为0.1mol/L的二甲胂酸钠缓冲液。 8(几种常用的不同pH值缓冲液的配制表 (1)0.2mol/L磷酸盐缓冲液(pH5.7,8.0) pH 0.2mol/L NaH2PO4(ml) 0.2mol/L Na2HPO4(ml) 5.7 93.5 6.5 5.8 92.0 8.0 5.9 90.0 10.0 6.0 87.7 12.3 6.1 85.0 15.0 6.2 81.5 18.5 6.3 77.5 22.5 6.4 73.5 26.5 6.5 68.5 31.5 6.6 62.5 37.5 6.7 56.5 43.5 6.8 51.0 49.0 6.9 45.0 55.0 7.0 39.0 61.0 7.1 33.0 67.0 7.2 28.0 72.0 7.3 23.0 67.0 7.4 19.0 81.0 7.5 16.0 84.0 7.6 13.0 87.0 7.7 10.5 89.5 7.8 8.5 91.5 7.9 7.0 93.0 8.0 5.3 94.7 (2)0.05mol/L Tris-HCl缓冲液(pH7.19,9.10) pH 0.2mol/L Tris(ml) 0.2mol/L HCl(ml) H2O 7.19 10.0 18.0 12.0 7.36 10.0 17.0 13.0 7.54 10.0 16.0 14.0 7.66 10.0 14.0 15.0 7.77 10.0 14.0 16.0 7.87 10.0 13.0 17.0 7.96 10.0 12.0 18.0 8.05 10.0 11.0 19.0 8.14 10.0 10.0 20.0 8.23 10.0 9.0 21.0 8.32 10.0 8.0 22.0 8.41 10.0 7.0 23.0 8.51 10.0 6.0 24.0 8.62 10.0 5.0 25.0 8.74 10.0 4.0 26.0 8.92 10.0 3.0 27.0 9.10 10.0 2.0 28.0 (3)0.2mol/L醋酸盐缓冲液(pH3.6,5.6) pH 0.2mol/L醋酸(ml) 0.2mol/L醋酸钠(ml) 3.6 46.3 3.7 3.8 44.0 6.0 4.0 41.0 9.0 4.2 36.8 13.2 4.4 30.5 19.5 4.6 25.5 24.5 4.8 20.0 30.0 5.0 14.8 35.2 5.2 10.5 39.5 5.4 8.8 41.2 5.6 4.8 45.2 (4)0.1mol/L二甲胂酸钠缓冲液(pH5.0,7.4) 0.2mol/L二甲胂酸钠pH 0.2N HCl(ml) 蒸馏水 (ml) 5.0 25 23.5 51.5 5.2 25 22.5 52.5 5.4 25 21.5 53.5 5.6 25 19.6 55.5 5.8 25 17.4 57.5 6.0 25 14.8 60.3 6.2 25 11.9 63.1 6.4 25 9.2 65.8 6.6 25 6.7 68.3 6.8 25 4.7 70.3 7.0 25 3.2 71.8 7.2 25 2.1 72.9 7.4 25 1.4 73.6 注:HCI可由NCO3代替，配制成二甲胂酸钠-HNO3缓冲液。 (5)0.2mol/L碳酸盐缓冲液(pH9.2,10.7) pH 0.2mol/L Na2CO3(ml) 0.2mol/L NaHCO3(ml) 9.2 4.0 46.0 9.3 7.5 42.5 9.4 9.5 40.5 9.5 13.0 37.0 9.6 16.0 34.0 9.7 19.5 30.5 9.8 22.0 28.0 9.9 25.0 25.0 10.0 27.5 22.5 10.1 30.0 20.0 10.2 33.0 17.0 10.3 35.5 14.5 10.4 38.5 11.5 10.5 40.5 9.5 10.6 42.5 7.5 10.7 45.0 5.0 三、显色液 免疫细胞化学中，由于抗原-抗体反应所形成的复合物本身无色，无法直接观察，因而需借助于某些化学基团的呈色作用，使其得以显示，以利于在显微镜下观察。常用的显色液有: 1(DAB(Diaminobenzidine)显色液 DAB即3，3-二氨基苯联胺 试剂:DAB(常用四盐酸盐) 50mg 0.05mol/L TB 100ml 30% H2O2 30,40μl 配制方法:先以少量0.05mol/L(pH7.6)的TB溶解DAB，然后加入余量TB，充分摇匀，使DAB终浓度为0.05%,过滤后显色前加入30%的H2O230,40μl，使其终浓度为0.01%。 DAB显色液主要用于免疫过氧化物酶法(如酶标法、PAP法等)，其终产物可直接在光镜下观察，也可经OsO4处理后，增加反应产物的电子密度，用于电镜观察。但有几点需注意:DAB溶解要完全，否则未溶解的颗粒沉积于标本上影响观察;DAB浓度不易过高，否则显色液呈棕色，增加背景染色，另外DAB有致癌作用，故操作时应戴手套，尽量避免与皮肤接触，用后及时彻底冲洗，接触DAB的实验用品最好经洗液浸泡24h后使用。 2(4-氯-1-萘酚(4-Cl-1-Naphthol)显色液 配方1:4-Cl-1-萘酚 100mg 纯乙醇 10ml 0.05mol/L TB(pH7.6) 190ml 30% H2O2 10μl(0.003%) 配制方法:先将4-Cl-1-萘酚溶解于乙醇中，然后加入Tb 19ml，用前加入30% H2O2使其终浓度为0.005%，切片显色时间通常为5,20min。 配方2:4-Cl-1-萘酚 N-二甲基甲酰胺(DMF) 0.05mol/L TB(pH7.6) 30% H2O2 配制方法:先将4-Cl-1-萘酚加入DMF中，加热溶解使呈乳白色，再加入TB，乳白色变为絮状，在7.5?加热5min后加入H2O2，搅动使絮状消失，趁热过滤，当降至略低于50?时才放入组织标本(注意:温度过高易损伤标本，过低则易重新出现沉淀)。显色时间通常为5min左右。 4-Cl-1-萘酚的终产物显示蓝色。 多数认为最好去除白色沉淀(如上述)，但Larsson等认为，白色沉淀可作为背景，使阳性部位更易观察。由于酒精可溶解4-Cl-1-萘酚显色的组织标本，勿用酒精脱水。 3(3-氨基-9-乙基卡唑(3-amino-9-ethylcarbozole,AEC)显色液 试剂:AEC 20mg 二甲基甲酰胺(DMF) 2.5ml 0.05mol/L醋酸缓冲液(pH5.5) 50ml 30%H2O2 25ml 配制方法:先将AEC溶于DMF中，再加入醋酸缓冲液充分混匀。临显色前，加入30%H2O2。切片显色时间为5,20min。 该显色液作用后，阳性部分呈深红色，加以苏木精或亮绿等作为背景染色，则效果更佳。由于终产物溶于酒精和水，故需用甘油封固。 4(TMB显色液 试剂:TMB HCl 亚硝基铁氰化钾 无水酒精 配制方法: (1)醋酸盐缓冲液:取1.0mol/L的HCl 190ml加入1.0mol/L的醋酸钠400ml中混合，再加蒸馏水稀释至1000ml，用醋酸或NaOH将pH调至3.3。 (2)A液:取上述缓冲液5ml，溶解100mg亚硝基铁氰化钾，加蒸馏水92.5ml混合。 (3)B液:称取5mg TMB加入2.5ml无水酒精中，可加热至37?,40?直到TMB完全溶解。 (4)孵育液:放入标本前数秒，取2.5ml B液及97.5ml A溶于试管中充分混合。(液体在20min内应保持清亮的黄绿色，否则可能已有污染)。酶反应时，加入终浓度为0.005%的H2O2。 (5)主要显色步骤:组织标本在蒸馏水(或PBS)中漂洗数次(每次约10,15min)后放入未加H2O2的孵育液中作用20min(19?,30?)，然后向孵育液中放入H2O2(每100ml孵育液中加0.3%的H2O2 1.0,5.0ml)继续孵育液20min左右(19?,23?)，捞出标本漂洗数次(共30min左右)。在0,4?条件下可在漂洗液放置4h直至贴片、脱水，封片。也可在贴片前在1%的中性红中负染2,3min，也可在1%派诺宁(pH3.3,3.5)中负染5min后贴片、脱水、封片。 TMB即四甲基联苯胺(Tetrabenzidine)是一种脂溶性较强的基团，因此容易进入细胞与细胞器中的HRP反应，且由于这种高度的脂溶性，使其易形成多聚体，在HRP活性部位产生粗大的、深兰色沉淀物，这使得TMB成为组化实验中的一种很好的发色团。同时反应产物的沉淀，使得HRP的活性部位更加暴露，利于酶氧化反应进行。TMB的反应产物为深蓝色，利于光镜观察，且反应产物越聚越大，常超出单个细胞器的范围(而DAB则被限制在其内)，故TMB反应的检测阈较低。由于上述优点，目前TMB常用于光镜及超微结构水平的HRP及HRP-WGA神经投射的研究。需要注意的是:TMB显色液中的A液和B液应在2h内新鲜配制。另外，TMB是一种较强的皮肤刺激剂，并有致癌的潜在可能，故使用时应带手套及在通风条件下操作。 5(NBT显色液 试剂A液:5%NBT:称 取0.5gNBT溶于10ml 70%的DMF(二甲基甲胺)内，充分混合，常存于4?，也可装成小份，-20?保存，用前复温。 B液:5% BCIP:称取BCIp 0.5g溶于10ml 100%的DMF内，混匀。4?或分装存于-20?，用前恢复至室温。 C(显色液:取A液40μl，加入到盛有10ml的0.1mol/l Tris-HCl(pH9.5,钤0.1mol/L NaCl、5mmol/L MgCl2)管内，充分混匀;再加入B液40μl，轻轻混合即可。最好用前新鲜配制。 NBT即四唑氮蓝(Nitro-Blue-Tetrazolium),MW=818,为深蓝色无定形微溶物质。BCIP即5-溴-4-氯-3-吲哚-磷酸盐(5-bromo-4-chloro-3-indolyl-phosphate)。当二者存在时，在碱性磷酸酶(Alkaline phosphatase, AP)作用下，NBT被还原形成显微镜下可见的蓝色或紫色沉淀。 四、粘附剂 在免疫细胞化学工作中，由于标本(如切片)的脱落常影响工作的质量的速度，故粘附剂的选择和使用就显得较为重要。 1(铬矾明胶液 试剂:铬矾 0.5g 明胶(Gelatine) 5g H2O ~1000ml 方法:在1000ml的烧杯或烧瓶中，以500,800ml H2O加温溶解明胶，待其完全溶解后，再加入铬矾。注意温度过高易使明胶烧糊，包被玻片时最好控制水温在70?。如有明显残渣，过滤后使用。 2(甲醛-明胶液 试剂:40%甲醛 2.5ml 明胶 0.5g 蒸馏水 至100ml 配制方法:用少许蒸馏水(约80ml)加热溶解明胶，待完全溶化后，加入甲醛，最后补充蒸馏水至100ml混匀即可。 3(多聚赖氨酸(Poly-L-Lycine,PLL) 试剂:多聚赖氧酸 5g 蒸馏水 ~1000ml 配制方法:称取PLL，溶于H2O，充分混合即可，此液浓度为0.5%，可适当稀释配成0.01,0.5%浓度。4?保存，也可-20?备用。PLL可反复冰冻，效果无明显影响，工作液常再稀释10,50倍。 4(Vectabond试剂 该试剂是Vector公司新进推出的一种新型玻片粘附剂。该试剂与一般的粘附剂不同，它是通过对玻璃表面起化学修饰作用，改变其表面的化学物理特性，使组织切片牢固地贴于玻璃片上，贴上后不易脱落，保留时间较久。一个试剂盒(7ml)可配成350ml工作液(用丙酮配)。处理载玻片前(事先酸处理)，用染色缸装好各种液体，按下列程序进行: 干净载玻片?丙酮5min?Vectabond试剂工作液(7ml Vectabond+ 350ml丙酮):将载玻片用镊子夹住浸入Vectabond试剂1,2次?dH2O，2×5min?干燥(37?过夜)?用铝箔包好，室温存放备用。 注意:避免污染(尘埃等)。 综上述方法处理的载片一般可存放半年以上。 五、封固剂 1(甘油-TBS及甘油-PBS 配制方法:按比例将甘油和TBS(或PBS)充分混合后，置4?，冰箱静止;待气泡排除后方可使用。 2(甘油-明胶(冻) 试剂:明胶 10g 甘油 12ml 蒸馏水 100ml 香草酚 少许 配制方法:称取1g明胶于温热(约40?)的蒸馏水中，充分溶解后过滤，再加入12ml甘油混合均匀。少许香草酚是为了防腐。 3(液体石蜡 液体石蜡因含杂质少，很少引起非特异性荧光，故常用于荧光组化及免疫荧光法时标本的封固。 4(DPX 试剂:Distrene 10g 酸二丁 5ml 二甲苯 35ml DPX为中性封固剂，用于多种染色方法匀不易褪色，但组织收缩较明显，故应尽量使其为均匀的一薄层。DPX现有商品出售，可直接应用。若感过于粘稠，也可加少量二甲苯稀释后应用。注意:二甲苯不可加得太多，二甲苯挥发后，片子上出现许多干燥的空泡，影响观察，遇有这种情况，可用二甲苯浸泡掉盖玻后重新封固。 六、酶消化液 1(0.1%胰蛋白酶 试剂:胰蛋白酶 0.1gm 0.1%氯化钙(pH7.8) 100ml 配制方法:先配制0.1%的CaCl2,用0.1mol/L的NaOH将其pH调至7.8，然后加入蛋白酶溶解之。用前将胰蛋白酶消化液在水浴中予热至37?(载有标本的玻片也在TBS中予热至同样样温度)。该消化液时间约为5,30min。 2(0.4%胃蛋白酶 试剂:胃蛋白酶 400mg 0.1N HCl 100ml 配制方法:同胰蛋白酶。消化时间在37?约为30min。 3(0.06% Pronase 试剂:Pronase 0.06g 0.05mol/L TB(pH7.5) 100ml 配制方法:同前。 在免疫细胞化学染色中，有时经Formalin过度固定的标本，常会产生过量的醛基，遮盖抗原，影响一抗与抗原的结合。用蛋白酶溶液消化，可起到暴露抗原部分的作用。消化时间应根据不同组织而异，总之，在保持组织形态不被破坏的前提下，宜尽量延长消化时间。以上三种酶消化液中，以第一种最为常用。 七、其它 1(蔗糖溶液 免疫细胞化学中应用的蔗精，常用浓度为5%,30%。一般光镜研究，仅用20%蔗糖处理已足矣，若制备电镜标本，在冰冻前最好经上行蔗糖(5%、10%、15%、20%及20%蔗糖-5%甘油等)处理，以确保其良好的细微结构。 (1)20%蔗糖液: 试剂:蔗糖 20g 0.1mol/L PB(pH7.5) 至100ml 配制方法:先以少许0.1mol/L的PB溶解蔗糖，再加0.1mol/l PB至100ml充分混合，置4?冰箱保存。 该液多用于纯光镜研究。标本在刚放入浓度如此高的蔗糖液，常浮在上面，当标本沉到底部时即可。通常光镜标本浸泡在20%蔗糖液中过夜。都能达到要求。 (2)20%蔗糖-5%甘油: 试剂:蔗糖 20g 甘油 5ml 0.1mol/L PB 至100ml(约95ml) 配制方法:先用少许PB溶解蔗糖后，再加入甘油，充分混匀，最后补足PB至100ml，于4?保存备用。 该液主要用于电镜标本的处理，常浸泡过夜(其它浓度的蔗糖中常分别为2h左右)。 蔗糖是一种廉价的防冻剂，兼有脱水的作用，它可减小标本在冰冻切片时冰晶形成的数量和大小，应用较 免疫组化步骤 1。4%多聚甲醛常规灌注固定，取材并置20%蔗糖溶液(4?)中过夜。蜡块制作。切片，贴片。0.01MKPBS清洗5min×3后; 2。加入配好的0.3%的过氧化氢甲醇溶液(甲醇80ml，0.01MKPBS 100ml，30%过氧化氢)30min，以消除内源性过氧化物酶的影响，0.01MPBS清洗5min×3; 3。加入配好的0.3% Triton X100(30% Triton X100，0.01MKPBS 100ml)30min，以增加细胞的通透性，0.01MKPBS清洗5min×3; 4。加入用血清稀释液(牛血清白蛋白1.00g，0.01MPBS 100ml，叠氮纳0.08g)稀释的一抗，4?存放24-48h;吸去抗体，0.01MKPBS洗5min×3; 5。加入0.01MKPBS稀释的的二抗，室温孵育2h。0.01MKPBS洗5min×3; 6。加入ABC复合物之类的抗体，室温孵育2h，0.01MKPBS洗5min×3;蒸馏水迅速冲三次; 7。加入显色液，进行免疫组织化学显色，时间一般不超过30min，可不时在显微镜下进行观察，待细胞着色而背底颜色较淡时马上吸去显色液，用蒸馏水迅速冲三次后加入0.01MKPBS终止反应; 8。梯度酒精脱水之后，封片，拍照。 免疫组化SP法步骤 admin SP(streptavidin-perosidase)法,即链霉菌抗生物素蛋白-过氧化物酶连结法. 按标记物质的种类，如荧光染料、放射性同位素、酶(主要有辣根过氧化物酶和碱性磷酸酶)、铁蛋白、胶体金等，可分为免疫荧光法、放射免疫法、酶标法和免疫金银法等。按染色步骤可分为直接法(又称一步法)和间接法(二步、三步或多步法);按结合方式可分为抗原—抗体结合，如过氧化物酶-抗过氧化物酶(PAP)法和亲和连接，如卵白素-生物素-过氧化物酶复合物(ABC)法、链霉菌抗生物素蛋白-过氧化物酶连结(SP)法等，其中SP法是最常使用的方法。 一般的sp法步骤啊,具体如下: 1.烤片，68?，20分钟, 2. 常规二甲苯脱蜡，梯度酒精脱水;二甲苯I 20min --二甲苯II 20 min--100%酒精I 10min --100%II 10min --95% 5min-- 80% 5min-- 70% 5min 3.阻断 灭活内源性过氧化物酶:3%H2O2 37?孵育10min，PBS冲洗3X5min; 4. 抗原修复:置0.01M枸橼酸缓冲液(PH 6.0)中用煮沸(95?，15,20min)，自然冷却20min 以上，再用冷水冲洗缸子，加快冷却至室温，PBS冲洗3X5min。 5. 正常羊血清工作液封闭，37?10 min，倾去勿洗. 6. 滴加一抗4? 冰箱孵育过夜，PBS冲洗3X5min(用PBS缓冲液代替一抗作阴性对照); 滴加生物素标记二抗, 37?孵育30min, PBS冲洗3X5min; 7. 滴加辣根过氧化物酶标记的链霉素卵白素工作液，37?孵育30min, PBS冲洗3X5min; 8. DAB/ H2O2反应染色，自来水充分冲洗后， 苏木素复染，常规脱水，透明，干燥，封片。 免疫组化(Elivison二步法) admin (1)石蜡切片置于67?烘箱中，烘片2小时，脱蜡至水，用pH7.4的PBS冲洗三次，每次3分钟(3×3’)。 (2)取一定量pH=6.0柠檬酸盐缓冲液，加入微波盒中，微波加热至沸腾，将脱蜡水化后的组织切片置于耐高温塑料切片架上，放入已沸腾的缓冲液中，中档微波处理10分钟，取出微波盒流水自然泠却，从缓冲液中取出玻片，先用蒸馏水冲洗两次，之后用PBS冲洗2×3’。(注:不是所有的抗体都需要微波修复的，视具体情况而定) (3)每张切片加1滴3% H2O2，室温下孵育10分钟，以阻断内源性过氧化物酶的活性。PBS冲洗3×3’。 (4)除去PBS液，每张切片加1滴相应的第一抗体(相应稀释倍数)，室温下孵育2小时。 (5)PBS冲洗3×5’。除去PBS液，每张切片加1滴聚合物增强剂(试剂A)，室温下孵育20分钟。PBS冲洗3×3’。 (6)除去PBS液，每张切片加1滴酶标抗鼠/兔聚合物(试剂B)，室温下孵育30分钟。PBS冲洗3×5’ (7)除去PBS液，每张切片加1滴新鲜配制的DAB液，显微镜下观察5分钟。 (8)苏木素复染，0.1%HCl分化，自来水冲洗，蓝化，切片经梯度酒精脱水干燥，二甲苯透明，中性树胶封固，晾干后观察。 注:即用型第二代免疫组化Elivision plus广谱试剂盒，购自福州脉新生物技术开发有限公司。包括:生物素化抗兔二抗、亲和素(Reagent A)、生物素,辣根过氧化物酶(Reagent B)、二氨基联苯胺(DAB) 免疫组化的经验(2),操作规程 jss2004 (一)、仪器设备 1)18cm不锈钢高压锅或电炉或医用微波炉; 2)水浴锅 (二)、试剂 1)PBS缓冲液(pH7.2,7.4):NaCl 137mmol/L，KCl 2.7mmol/L，Na2HPO4 4.3mmol/L，KH2PO4 1.4mmol/L。 2)0.01mol/L柠檬酸盐缓冲液(CB，pH6.0，1000ml):柠檬酸三钠 3g，柠檬酸 0.4g。 3)0.5mol/L EDTA缓冲液(pH8.0):700ml水中溶解186.1gEDTA?2H2O，用10 mmol/L NaOH 调至pH8.0,加水至1000ml。 4)1mol/L的TBS缓冲液(pH8.0):在800ml水中溶解121gTris碱，用1N的HCl调至pH8.0,加水至1000ml。 5)酶消化液: a、0.1%胰蛋白酶液:用0.1%CaCl2(pH7.8) 配制。 b、0.4%胃蛋白酶液:用0.1N的HCl配制。 6)3%甲醇-H2O2溶液:用30%H2O2和80%甲醇溶液配制。 7)封裱剂: a、甘油和0.5mmol/L碳酸盐缓冲液(pH9.0,9.5)等量混合; b、油和TBS(或PBS)配制。 8)TBS/PBS pH9.0,9.5，适用于荧光显微镜标本;pH7.0,7.4适合于光学显微镜标本。 (三)、操作流程 1、脱蜡和水化 脱蜡前，应将组织芯片在室温中放置60分钟或60?恒温箱中烘烤20分钟。 1)组织芯片置于二甲苯中浸泡10分钟，更换二甲苯后再浸泡10分钟; 2)无水乙醇中浸泡5分钟; 3)95%乙醇中浸泡5分钟; 4)70%乙醇中浸泡5分钟; 2、抗原修复 用于福尔马林固定的石蜡包埋组织芯片。 1)抗原热修复 (1)高压热修复 在沸水中加入EDTA(pH8.0)或0.01M枸橼酸钠缓冲溶液(pH6.0)。盖上不锈钢高压锅的盖子，但不进行锁定。将玻片置于金属染色架上，缓慢加压，使玻片在缓冲液中浸泡5分钟，然后将盖子锁定，小阀门将会升起来。10分钟后，去除热源，置入凉水中，当小阀门沉下去后打开盖子。本方法适用于较难检测或核抗原的抗原修复。 (2)煮沸热修复 电炉或者水浴锅加热0.01M枸橼酸钠缓冲溶液(pH6.0)至95?左右，放入组织芯片加热10~15分钟。 (3)微波热修复 在微波炉里加热0.01M枸橼酸钠缓冲溶液(pH6.0)至沸腾后将组织芯片放入，断电，间隔5~10分钟，反复1-2次。适用的抗原有:AR，Bax，Bcl-2，C-fos，X-jun，C-kit，C-myc，E-cadherin，Chromogranin A，Cyclin，ER，Heat shock protein，HPV，Ki-67，MDMZ，p53，p34，p16，p15，P-glycoprotein，PKC，PR，PCNA，ras，Rb，Topoismerase?等。是以高温，高压对常规固定的石蜡切片进行抗原修复或复原的一种非蛋白酶消化以提高抗原抗体阳性检测的一种方法和技术手段。甲醛和蛋白水解交联过程中氨基酸侧链上的某些基团(抗原决定簇)如咪唑、吲哚等基团受到影响，通过120?高温或强碱处理后，可使交联打开。 2)酶消化方法 常用0.1%胰蛋白酶和0.4%胃蛋白酶液。胰蛋白酶使用前预热至37?，切片也预热至37?，消化时间约为5~30分钟;胃蛋白酶消化37?时间为30分钟。适用于被固定遮避的抗原，其中有:Collagen，Complement，Cytokeratin，C-erB-2，GFAP，LCA，LN等。 3、免疫组织化学染色 SP法 1)脱蜡、水化; 2)PBS洗2,3次各5分钟; 3)3%H2O2(80%甲醇)滴加在TMA上，室温静置10分钟; 4)PBS洗2,3次各5分钟; 5)抗原修复; 6)PBS洗2,3次各5分钟; 7)滴加正常山羊血清封闭液,室温20分钟。甩去多余液体。 8)滴加?抗50μl，室温静置1小时或者4?过夜或者37?1小时。 9)4?过夜后需在37?复温45分钟。 10)PBS洗3次各5分钟; 11)滴加?抗40,50μl，室温静置，或37?1小时; 12)II抗中可加入0.05%的tween-20。 13)PBS洗3次各5分钟; 14)DAB显色5,10分钟，在显微镜下掌握染色程度; 15)PBS或自来水冲洗10分钟; 16)苏木精复染2分钟，盐酸酒精分化; 17)自来水冲洗10,15分钟; 18)脱水、透明、封片、镜检。 SABC法 1)脱蜡、水化。 2)PBS洗两次各5分钟。 3)用蒸馏水或PBS配置新鲜的3%H2O2，室温封闭5,10分钟，蒸馏水洗3次。 4)抗原修复。 5)PBS洗5分钟。 6)滴加正常山羊血清封闭液，室温20分钟。甩去多余液体。 7)滴加?抗,室温1小时或者4?过夜或者37?1小时(4?过夜后在37?复温45分钟)。 8)PBS洗三次每次2分钟。 9)滴加生物素化二抗,20?,37?20分钟。 10)PBC洗3次每次2分钟。 11)滴加试剂SABC，20?,37?20分钟。 12)PBS洗4次每次5分钟。 13)DAB显色:DAB显色试剂盒或者自配显色剂显色(镜下掌握显色程度)。 14)蒸馏水洗。苏木素复染2分钟、盐酸酒精分化。 15)脱水、透明、封片、镜检。