Nakagawa Takenobu, Ohnishi Koji, Kosaki Yui, Saito Yoichi, Horlad Hasita, Fujiwara Yukio, Takeya Motohiro, Komohara Yoshihiro
Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University.
J Clin Exp Hematop. 2017;57(1):31-36. doi: 10.3960/jslrt.17017.
Macrophages are closely related to various diseases and it is therefore important that the properties of macrophages are adequately evaluated in human diseases and mouse disease models. Immunohistochemistry (IHC) of formalin fixed paraffin-embedded (FFPE) samples is a very useful tool for examination of macrophages; however, an adequate IHC protocol is required for the examination of macrophage states. In this study, we assessed various antigen retrieval methods in order to devise the optimal protocols for staining of macrophages with a range of antibodies. Optimum combinations of primary antibodies and antigen retrieval protocols were determined; for example, heat treatment with ethylenediamine tetraacetic acid solution, pH 8.0, was the best procedure for IHC using mouse anti-Iba1 and human anti-CD11b, -CD163, -CD169, -CD204, and -CD206 antibodies. Moreover, we found that the immunoreactivity of sliced tissue sections decreased gradually over time in long term storage but that this immunoreactivity was preserved in storage at -80 °C in a deep freezer. The optimal IHC protocols and storage procedures that were determined in this study should be a useful tool for macrophage research.
巨噬细胞与多种疾病密切相关，因此在人类疾病和小鼠疾病模型中充分评估巨噬细胞的特性非常重要。福尔马林固定石蜡包埋（FFPE）样本的免疫组织化学（IHC）是检测巨噬细胞的一种非常有用的工具；然而，检测巨噬细胞状态需要适当的免疫组化方案。在本研究中，我们评估了各种抗原修复方法，以设计出用一系列抗体对巨噬细胞进行染色的最佳方案。确定了一抗和抗原修复方案的最佳组合；例如，使用pH 8.0的乙二胺四乙酸溶液进行热处理，是使用小鼠抗Iba1和人抗CD11b、-CD163、-CD169、-CD204和-CD206抗体进行免疫组化的最佳方法。此外，我们发现，切片组织在长期储存过程中免疫反应性会逐渐下降，但在-80°C的深冻冰箱中储存时，这种免疫反应性得以保留。本研究确定的最佳免疫组化方案和储存程序应成为巨噬细胞研究的有用工具。
