Background

Aldehyde fixation is a common process used to preserve the complex structure of biological samples ex vivo. This method of fixation relies on the formation of covalent bonds between aldehydes and amines present in the biomolecules of the sample. Aldehyde fixation is routinely performed in histological studies, however fixed tissue samples are rarely used for non-histological purposes as the fixation process is thought to make brain tissue unsuitable for traditional proteomic analyses such as Western blot. Advances in antigen-retrieval procedures have allowed detectable levels of protein to be solubilized from formaldehyde fixed tissue, opening the door for aldehyde-fixed samples to be used in both histological and proteomic approaches.

New method

Here, we developed a series of antigen-retrieval steps for use on fixed-brain lysates to make them suitable for analysis by Western blot.

Results

Prolonged exposure of the tissue homogenate to high temperature (90 °C for 2 h) in the presence of a concentrated formaldehyde scavenger and ionic detergent was sufficient to reveal a variety of synaptic and non-synaptic proteins on membrane blots.

Conclusion

This protocol has significant utility for future studies using fixed tissue samples in a variety of neuropathological conditions.

中文翻译：

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释放大脑：从固定的大脑组织中检测蛋白质印迹蛋白的新方法

背景

醛固定是用于保留离体生物样品复杂结构的常用方法。这种固定方法依赖于样品生物分子中存在的醛和胺之间的共价键的形成。醛固定在组织学研究中是常规进行的，但是固定的组织样本很少用于非组织学目的，因为固定过程被认为会使脑组织不适合传统的蛋白质组学分析，例如蛋白质印迹。抗原回收程序的进步已使可检测水平的蛋白质从甲醛固定的组织中溶解，从而为用于组织学和蛋白质组学方法的醛固定样品打开了大门。

新方法

在这里，我们开发了一系列针对固定脑裂解物的抗原回收步骤，以使其适合通过蛋白质印迹法进行分析。

结果

在浓缩甲醛清除剂和离子去污剂存在下，组织匀浆长时间暴露于高温（90°C 2小时）足以在膜印迹上揭示各种突触和非突触蛋白。

结论

该协议对于在各种神经病理状况下使用固定组织样本的未来研究具有重要的实用性。