A new simplified catalyzed signal amplification system for minimizing non-specific staining in tissues with supersensitive immunohistochemistry
A new simplified catalyzed signal amplification system for minimizing non-specific staining in tissues with supersensitive immunohistochemistry
复制标题
DOI：
10.1679/aohc.68.1
复制
发表时间：
2005-01-01
影响因子：
--
通讯作者：
Murata, F
中科院分区：
文献类型：
--
作者：
Hasui, K;Murata, F
我们研究了催化报告细胞沉积（CARD）反应中的非特异性染色，并基于我们简化的催化信号放大（CSA）系统改进了其在超敏感免疫组织化学中的阻断方法（Hasui et al. 2002）。在使用生物素酪酰胺的CARD反应中，用酪蛋白溶液或3%牛血清白蛋白(BSA)-磷酸盐缓冲盐水（PBS） （0.1% Tween 20）预处理可以减少非特异性染色。在使用fitc标记的酪酰胺进行CARD反应时，可以用0.3% BSA-PBS加0.1% Tween 20或3%聚乙二醇- pbs加01% Tween 20预处理来阻断非特异性染色。因此，我们的新简化CSA体系具有以下特点：1)破坏内源性过氧化物酶活性；2)阻断一抗的非特异性反应；3)一抗反应；4)酪蛋白处理阻断聚合物试剂的非特异性反应；5)聚合物反应；6)酪蛋白处理阻断CARD反应的非特异性反应；7) CARD反应；8)沉积酪胺的检测。事实证明，这种新系统可用于检测内源性富含生物素的组织（如胃肠道和肝脏）中极少量的抗原。通过这种方法，可以检测到G1期细胞周期中的Ki67抗原，并且Ki67抗原的代谢紊乱与胃类癌有关。我们相信这种新的简化CSA系统代表了光显微镜研究中超敏感免疫组织化学的新标准。
We investigated non-specific staining in a catalyzed reporter deposition (CARD) reaction and improved its blocking methods in supersensitive immunohistochemistry, based on our simplified catalyzed signal amplification (CSA) system (Hasui et al. 2002). In the CARD reaction using biotinyl tyramide, non-specific staining could be reduced by pretreatment with a casein solution or 3% bovine serum albumin (BSA)-phosphate buffer saline (PBS) with 0.1% Tween 20. In the CARD reaction using FITC-labeled tyramide, non-specific staining could be blocked by pretreatment with 0.3% BSA-PBS with 0.1% Tween 20 or 3% polyethylene glycol-PBS with 01% Tween 20. Thus, our new simplified CSA system features: 1) destruction of the endogenous peroxidase activity; 2) blocking of the non-specific reaction of the primary antibody; 3) a primary antibody reaction; 4) blocking of the non-specific reaction of the polymer reagent by casein treatment; 5) a polymer reaction; 6) blocking of the nonspecific reaction of CARD reaction by casein treatment; 7) a CARD reaction; and 8) detection of deposited tyramide. This new system proved useful for detecting an extremely low amount of antigen in the endogenous biotin-rich tissues such as the gastrointestinal tract and liver. By this method, the Ki67 antigen in the G1 phase cell cycle could be detected and a metabolic disorder of the Ki67 antigen was implicated in a carcinoid tumor in the stomach. We believe that this new simplified CSA system represents a new standard of supersensitive immunohistochemistry for use in light-microscopic investigation.