Department of Genetics and Pathology, Uppsala University, Sweden.
J Histochem Cytochem. 2010 Mar;58(3):237-46. doi: 10.1369/jhc.2009.954321. Epub 2009 Nov 9.
Pathology archives harbor large amounts of formalin-fixed, paraffin-embedded tissue samples, used mainly in clinical diagnostics but also for research purposes. Introduction of heat-induced antigen retrieval has enabled the use of tissue samples for extensive immunohistochemical analysis, despite the fact that antigen retrieval may not recover all epitopes, owing to alterations of the native protein structure induced by formalin. The aim of this study was to investigate how different fixatives influence protein recognition by immunodetection methods in tissues, cell preparations, and protein lysates, as compared with formalin. Seventy-two affinity-purified polyclonal antibodies were used to evaluate seven different fixatives. The aldehyde-based fixative Glyo-fixx proved to be excellent for preservation of proteins in tissue detected by immunohistochemistry (IHC), similar to formalin. A non-aldehyde-based fixative, NEO-FIX was superior for fixation of cultured cells, in regard to morphology, and thereby also advantageous for IHC. Large variability in the amount of protein extracted from the differently fixed tissues was observed, and the HOPE fixative provided the overall highest yield of protein. In conclusion, morphological resolution and immunoreactivity were superior in tissues fixed with aldehyde-based fixatives, whereas the use of non-aldehyde-based fixatives can be advantageous in obtaining high protein yield for Western blot analysis. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
病理档案库中储存着大量经福尔马林固定、石蜡包埋的组织样本，主要用于临床诊断，但也可用于研究目的。热诱导抗原修复的引入使得组织样本能够进行广泛的免疫组织化学分析，尽管抗原修复可能无法恢复所有表位，因为福尔马林诱导的天然蛋白质结构的改变。本研究旨在探讨与福尔马林相比，不同固定剂如何影响免疫检测方法在组织、细胞制剂和蛋白裂解物中对蛋白质的识别。使用 72 种亲和纯化的多克隆抗体来评估七种不同的固定剂。醛基固定剂 Glyo-fixx 被证明在组织中的蛋白质免疫组织化学（IHC）检测中具有优异的保存效果，类似于福尔马林。一种非醛基固定剂 NEO-FIX 在培养细胞的固定方面优于形态学，因此也有利于 IHC。从不同固定的组织中提取的蛋白质的量存在很大的差异，HOPE 固定剂提供了总体上最高的蛋白质产量。总之，基于醛的固定剂固定的组织在形态分辨率和免疫反应性方面表现更好，而使用非醛基固定剂在获得用于 Western blot 分析的高蛋白质产量方面可能具有优势。本文包含在线补充材料，可在 http://www.jhc.org 上查看。请访问本文的在线版本以查看这些材料。
