ANTIGEN UNMASKING ON FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE-SECTIONS
ANTIGEN UNMASKING ON FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE-SECTIONS
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DOI：
10.1002/path.1711710205
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发表时间：
1993-10-01
影响因子：
7.3
通讯作者：
RILKE, F
中科院分区：
文献类型：
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作者：
CATTORETTI, G;PILERI, S;RILKE, F
通过使用一组诊断相关抗体（抗细胞角蛋白、波形蛋白、S-100、T-和B-细胞受体、Ki-67/MIB 1、肌肉肌动蛋白），优化并比较了用于在福尔马林固定、石蜡包埋、脱蜡切片上抗原解蔽的酶促和非酶促处理。通过在微波炉中在0.01 M盐溶液（pH 6）或6 M尿素中煮沸载玻片，获得非酶促解蔽。胰蛋白酶或链霉蛋白酶消化用于比较，发现对于某些试剂是必要的。然后，研究扩展到256种抗体;其中48种的表位氨基酸序列是已知的。我们发现，酶和非酶抗原解蔽不依赖于表位序列，但一些抗原选择性地受益于一种治疗，而不是从其他。蛋白质变性是导致微波炉煮沸载玻片上免疫检测的可能机制;这一建议得到了变性溶液的使用以及观察到内源性酶被灭活和煮沸后一些抗原不再免疫检测的支持。非酶法抗原解蔽是一个强大的新工具，扩大使用的抗体免疫染色福尔马林固定，石蜡包埋切片，并应与传统的酶法平行使用。
Enzymatic and non-enzymatic treatments for antigen unmasking on formalin-fixed, paraffin-embedded, dewaxed sections were optimized and compared by the use of a panel of antibodies of diagnostic relevance (anti-cytokeratins, vimentin, S-100, T- and B-cell receptors, Ki-67/MIB 1, muscle actin). Non-enzymatic unmasking was obtained by boiling the slides in a microwave oven in 0.01 M salt solution (pH 6) or in 6 M urea. Trypsin or pronase digestion was used for comparison and found to be necessary for some of the reagents. The investigation was then extended to 256 antibodies; the epitopic amino acid sequence was known for 48 of them. We found that enzymatic and non-enzymatic antigen unmasking are not dependent on the epitope sequence, but some antigens benefit selectively from one treatment but not from the other. Denaturation of proteins is the likely mechanism which leads to immunodetection on microwave oven-boiled slides; this suggestion is supported by the use of denaturating solutions and by the observation that endogenous enzymes were inactivated and a few antigens were no longer immunodetectable after boiling. Non-enzymatic methods for antigen unmasking are a powerful new tool for broadening the use of antibodies for immunostaining formalin-fixed, paraffin-embedded sections and should be used in parallel with the traditional enzymatic methods.