免疫组化ABC法

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,免疫组化,ABC,法,组织抗原,第一抗体,第二抗体,HRP,生物素,亲和素,原理：,酶标记,Biotin,形成酶素化,Biotin,一,ABC,法,酶素化,Biotin,Avidin,-ABC,复合物,抗原,-,特异抗体,-Biotin,标记二抗,-,Avidin,Biotin HRP,实验原理,步骤：,三步法,优点：,敏感性较高,特异性强,应用范围广,缺点：,某些脏器存在内源性生物素干扰,Avidin,与糖基、纤维组织、核酸及细胞膜中 磷脂有非特异反应,实验步骤,一、切片和脱蜡,切,5-10,微米的薄片，贴于载玻片上，脱蜡后进行后续反应。步骤如下：,（,1,）二甲苯脱蜡,（,2,）梯度酒精水化（无水酒精、,95%,酒精、,90%,酒精、,85%,酒精、,80%,酒精）,二、微波修复抗原,免疫组化,ABC,法,实验步骤,三、,ABC,法,含,1%Triton X-100,和,0.03%H2O2,的,0.01 mol/L,磷酸盐缓冲液,(PBS,pH7.4),室温,15min,；,含,3%,牛血清白蛋白,(BSA,Sigma),和,1%,正常羊血清,(NGS,Vector),的,0.01 mol/L PBS,室温,30min,；,免疫组化,ABC,法,实验步骤,一抗室温孵育过夜。抗体用含,3%,牛血清白蛋白,(BSA,，,Sigma),和,1%NGS,的,0.01 mol/L PBS,稀释,(,下同,),；,0.01 mol/L PBS,漂洗,3,次，每次,5min;,生物素化二抗室温孵育,2h;,免疫组化,ABC,法,实验步骤,0.01 mol/L PBS,漂洗,3,次，每次,5min;ABC(1:100,Vector),室温,2h;,0.01 mol/L PBS,漂洗,3,次，每次,5min;,含,0.03%DAB,和,0.006%H2O2,的,Tris,盐酸缓冲液,(TB,pH7.6),室温,1015 min,。,免疫组化,ABC,法,实验步骤,（,10,）,0.01 mol/L PBS,漂洗,3,次，每次,5min;,（,11,）贴片，甘油封片,四、观察结果并分析,免疫组化,ABC,法,实验对照设置,免疫组化,ABC,法,对照染色的目的,-,排除假阴性和假阳性。,