肾组织冰冻切片中血红素氧合酶-1的免疫组织化学检测  

Immunohistochemical staining of heme oxygenase-1 in frozen kidney sections

作　　者：田恒宇 周汉新 

机构地区：[1]暨南大学第二临床医学院深圳市人民医院肝胆胰外科,深圳518020

出　　处：《中华实验外科杂志》2013年第9期1921-1923,F0003,F0004,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81260124）

摘　　要：目的 探讨大鼠肾脏组织冰冻切片中血红素氧合酶-1（HO-1）的免疫组织化学检测方法及其干扰因素.方法 腹腔内注射钴原卟啉（CoPP）诱导Wistar大鼠（n=24）肾脏高表达HO-1；比较常规链霉菌抗生物素蛋白-过氧化物酶（SP）法（n=8）、SP＋蛋清封闭法（n=8）和Dako EnVision二步法（n=8）消除非特异性染色的效果,并通过检测已知阳性组织的HO-1表达加以验证.结果 Western blot法检测证实CoPP成功诱导全部24只Wistar大鼠肾脏高表达HO-1.用免疫组织化学法检测HO-1在大鼠肾脏组织中的表达时,可见（1）正常大鼠肾组织仅部分大血管区有内源性过氧化物酶的着色,能被0.3％H202·甲醇或3％ H202消除；（2）正常大鼠肾组织有丰富的内源性生物素表达,常规SP法染色（n=8）出现较强的非特异性染色；SP＋蛋清封闭法染色（n=8）不能消除非特异性染色,反而有放大作用；Dako EnVision二步法（n=8）可以有效消除非特异性染色,良好显示HO-1在肾脏组织中的分布.结论 内源性生物素是冰冻切片免疫组织化学染色的主要干扰因素；常规SP法或加用蛋清封闭不能消除其干扰；EnVision二步法可以有效避免非特异性干扰,良好的显示HO-1在阳性肾脏组织中的表达.Objective To study the immunohistochemical staining of heme oxygenase-1 （HO-1）in frozen kidney sections and the interference factors.Methods High expression of HO-1 in the kidney of rats （n =24） was achieved through CoPP administration.The Avidin-Biotin Complex system （SP system）with （n =8） /without （n =8） egg white blocking was compared with the Dako EnVision system （n =8） in the staining of endogenous peroxydase,endogenous biotin and HO-1 in the frozen kidney sections of rats.Results CoPP induced HO-1 high expression in all 24 rat kidneys.There was no endogenous peroxydase in the normal rat kidneys,except for few area of large blood vessels,which could be blocked by 0.3％ H202 ·methanol or 3％ H202.Endogenous biotin was widely detected in frozen rat kidney sections,which could not be blocked by 20％ egg white and even be enlarged instead （n =8）.The Dako EnVision system could effectively avoid the the non-specific staining of the endogenous biotin activity and showed a clean strong expression of HO-1 （n =8）.Conclusion Endogenous biotin is the major interference factor of the immunohistochemical staining in frozen sections.The routine SP system with/without egg white blocking can not eliminate the interference.EnVision system gives an efficiency of the detection of HO-1 with a clean backgroud.

关 键 词：生物素 卵白素 免疫组织化学 大鼠 

分 类 号：R446.8[医药卫生—诊断学]