固定液及抗原修复液pH值对免疫组化染色的影响

目的 :研究固定液pH值和抗原修复液pH值对免疫组化染色结果的影响。方法 :取正常皮肤、胃粘膜、子宫肌瘤、乳腺癌组织 ,分别用 pH值 3.0 ,5 .0 ,7.0 ,8.0 ,10 .0的缓冲福尔马林固定液固定。选用EMA、SMA、ER、CK(AE1/AE3 )为一抗 ,Evinsion二步法免疫组化染色。染色时分别用 pH值 6 .0 ,7.0 ,8.0的抗原修复液或用市售pH值 8.0的EDTA作微波抗原修复。结果 :不同条件可造成阳性细胞数量及染色强度变化 ,本实验得知CK (AE1/AE3 )、ER是以阳性细胞数目改变为主 ,SMA、EMA是以染色强度改变为主。结论 :(1)使用 10 %中性或微碱性缓冲福尔马林固定液 ,固定效果最佳。 (2 )本实验所用的 4种组织 ,抗原修复液选择pH值 7.0、8.0均优于 pH值6 .0 ,皮肤组织选择EDTA(pH值 8.0 ) ,雌激素受体选择 pH值 8.0时染色效果最佳。 (3)固定液pH值 >8.0容易造成背景染色 ,修复液 >8.0影响切片附着 ,造成脱片

Effects of different pH values of fixatives and antigen retrieval buffers on immunohistochemical staining

Objective: To evaluate the influence of pH values of fixatives and antigen retrievial buffers on immunostains. Methods: Samples of normal skin,gastric mucosa,uteri leiomyoma and breast carcinoma were fixed in buffered formalin with different pH values, namely 3.0,5.0,7.0,8.0 and 10.0 to detect EMA,SMA,ER and CK(AE 1/AE 3) expression.Evinision two step method was employed.Antigen retrieval was achieved by microwave using sodium citrate solution with pH 6.0, 7.0, 8.0 or pH 8.0 EDTA solution respectively. Results: The number and intensity of positive cells varied due to different conditions.The change of positive cell's number is important for CK(AE 1/AE 3) expression,and the change of positive intensity is important for SMA and EMA expression.Conclusions: (1)The best results could be obtained by the use of 10% neutral or alkalescent buffered formalin.(2)Antigen retrieval effect is better in the solution of pH 7.0,8.0 than in that of pH 6.0. EDTA(pH 8.0) appears better for skin.The best effect of Estrogen Receptor needs pH 8.0 sodium citrate solution.(3)The background could be easily induced by the fixative over pH 8.0.Antigen retrieval solution over pH 8.0 could result in detachment.