热休克蛋白（HSPs）通常在所有有核细胞中表达，但它们的表达特点不同。具体而言，HSP90α和HSP90β具有高度相似的序列，并且尚未完全研究过它们作为分子伴侣的个体和补偿功能、客体蛋白的差异以及伴随exosome的细胞外分布。免疫组织化学染色是一种利用特异性抗体可视化目标抗原的存在和定位的技术，多重免疫染色法可以显示同一肿瘤组织中蛋白质表达的差异以及感兴趣蛋白质在肿瘤组织或单个细胞中的定位。常用的多重免疫染色法使用不同反应动物种属的多个二级抗体以识别和检测不同的抗原，因此需要使用不同的动物免疫原初抗体。此外，荧光抗体法是主要的多重染色方法，但其关键缺点是无法制备永久标本。在此，我们概述了一种基于酶抗体法的HSP90α和HSP90β的多重染色方法，允许制备永久标本而不受免疫动物种属的限制。© 2023年。作者（们）独家授权Springer Science+Business Media, LLC, Springer Nature的一部分。

Heat shock proteins (HSPs) are often expressed in all nucleated cells, but their expression profiles differ. In particular, HSP90α and HSP90β have high sequence identity and have not been fully examined for their individual and compensatory functions as molecular chaperones, differences in client proteins, and extracellular distributions with exosomes. Immunohistochemical staining is a technique to visualize the presence and localization of target antigens using specific antibodies, of which the multiplex immunostaining method can reveal differences in protein expression in the same tumor tissue and the localization of proteins of interest within tumor tissue or single cells. The common multiplex immunostaining method uses multiple secondary antibodies of different reacting animal species to identify and detect different antigens, thus requiring different animals to be immunized with each primary antibody. Furthermore, the fluorescent-antibody method is the predominant multiplex staining method but has the critical disadvantage that permanent specimens cannot be prepared. Here, we outline a multiplex staining method for HSP90α and HSP90β based on the enzyme-antibody method that allows permanent specimens to be prepared without the restriction of immunized animal species.© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.