肺腺癌肿瘤相关巨噬细胞的免疫组织化学双标染色方法优化  

Modification and improvement of immunohistochemical double staining for tumor associated macrophages in lung adenocarcinoma tissues

作　　者：张宪超[1] 张翔[1] 曹棉富[1] 陈露[1] 党微旗[1] 宋康[1] 崔有宏[1] 张厦[1] 卞修武[1] 

机构地区：[1]第三军医大学西南医院全军临床病理学研究所/西南癌症中心,重庆400038

出　　处：《第三军医大学学报》2017年第11期1155-1158,共4页Journal of Third Military Medical University

基　　金：国家重点研发计划重点专项(2016YFC201801);国家自然科学基金面上项目(81372273);西南医院生物强院科技创新计划(SWH2016JCZD-04)~~

摘　　要：目的通过对现有的免疫组织化学双重标记方法(双标)的改进,探索一种稳定且易于辨认的显色方法,便于在普通光学显微镜下观察肺腺癌组织中肿瘤相关巨噬细胞的分布情况。方法改进的免疫组织化学双标染色分别使用二抗链接的碱性磷酸酶和辣根过氧化物酶作为底物显色酶,底物分别选用固红(AP-Red)和二氨基联苯胺(DAB),在显微镜下次序控制双标显色,观察肺癌组织中肿瘤相关巨噬细胞的分布情况。结果免疫组织化学双重标记中第一标记AP-Red的红色与第二标记DAB的棕黄色反差大,背景低,易于分辨。两种颜色标记的同一部位呈现砖红色,易于表示双标部位。统计显示在肺腺癌组织标本中,以M2型肿瘤相关巨噬细胞为主,约占80%。结论改进后的显色系统建立了简便、可靠的免疫组织化学双重标记新方法。标记结果可以清晰稳定显示肿瘤相关巨噬细胞在肺腺癌组织中分布情况,便于进一步研究其在肺癌发生、发展中的作用。Objective To explore a stable and easy-to-identify color developing method by modifying immunohistochemical double-staining （D-IHC） so as to investigate the distribution of tumor associated macrophages （TAMs） in lung adenocarcinoma tissue. Methods Although as the same as the traditional double-enzyme methods which used the enzyme-conjugated secondary antibody to respectively link alkaline phosphatase （ALP） and horseradish peroxidase （HRP） as catalyze chromogenic substrates, AP-Red and diaminobenzidine （DAB） were chosen instead of 5-Bromo-4-Chloro-3-Indolyl Phosphate （BCIP/NBT） and 3- Amino-9-ethylcarbazole （AEC） as chromogenic substrates. The distribution of TAMs in lung cancer tissues was observed under the microscope after the double staining were controlled in order. Results As the image was highly contrast and easily distinguishable with low background under a light microscope, Fast Red （first labeling） and DAB （second labeling） were suitable for application in D-IHC. In addition, these 2 kinds of color labeling were merged as brick red at the same position, which was recognizable under a light microscope. The statistical results revealed that M2 type of TAMs were priority （ accounting for 80% ） in the lung adenocarcinoma tissue samples. Conclusion The modified method is simple and reliable with stable labeling results, and will be useful in the exploration of lung adenocarcinoma and other biomedical research.

关 键 词：免疫组织化学染色 辣根过氧化酶 碱性磷酸酶 肿瘤相关巨噬细胞 肺癌 

分 类 号：R392.12[医药卫生—免疫学] R446.8[医药卫生—基础医学]