Kobayashi Motohiro, Nakayama Jun
Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
Methods Enzymol. 2010;479:271-89. doi: 10.1016/S0076-6879(10)79016-9.
Over the last four decades, immunohistochemistry (IHC) has become an invaluable technique to detect antigens in tissue sections. Compared to Western blotting analysis, IHC is advantageous in determining histological distribution and localization of the antigen. Another advantage, if one can access human formalin-fixed paraffin-embedded (FFPE) blocks of disease tissues, is that IHC makes it possible to analyze diseases retrospectively from archived pathological tissue specimens. In this chapter, we describe protocols used for both conventional and multiple immunostainings using FFPE tissue sections, which have been used for quantitative analysis of high endothelial venule (HEV)-like vessels and lymphocyte subsets attached to HEV-like vessels in our studies of chronic inflammatory gastrointestinal diseases. We also describe in detail a protocol using an L-selectinIgM chimera in situ binding assay on FFPE tissue sections for functional detection of L-selectin ligand carbohydrates expressed on HEV-like vessels. After presenting each protocol, we provide practical examples for its use obtained from our studies.
在过去的四十年里,免疫组织化学(IHC)已成为检测组织切片中抗原的一项极为重要的技术。与蛋白质印迹分析相比,免疫组织化学在确定抗原的组织学分布和定位方面具有优势。另一个优势是,如果能够获取疾病组织的人福尔马林固定石蜡包埋(FFPE)块,免疫组织化学使得从存档的病理组织标本中回顾性分析疾病成为可能。在本章中,我们描述了使用FFPE组织切片进行传统免疫染色和多重免疫染色的方案,这些方案在我们对慢性炎症性胃肠疾病的研究中用于高内皮微静脉(HEV)样血管和附着于HEV样血管的淋巴细胞亚群的定量分析。我们还详细描述了一种在FFPE组织切片上使用L-选择素IgM嵌合体原位结合测定法来功能性检测HEV样血管上表达的L-选择素配体碳水化合物的方案。在介绍每个方案后,我们提供了从我们的研究中获得的其使用的实际示例。