免疫荧光双重染色的激光共聚焦显微镜样品制备及.pdfVIP

免 疫 学 杂 志 　第 23 卷 　第 3 期 　2007 年 5 月 　　IMMUNOLOGICAL JOURNAL Vol. 23 No. 3 May. 2007 3·44 · 　 ( ) [文章编号 2007 免疫荧光双重染色的激光共聚焦显微镜样品制备及观察 ( ) 路 　菊 ,孙 　玮 ,陈德英 第三军医大学高原军事医学系中心实验室 ,重庆 400038 [摘 　要] 　目的　详尽介绍免疫荧光双标染色方法 ,为激光共聚焦显微镜观察提供理想的样品。方法 　大鼠腹腔注射 Brdu ,40 gL 多聚甲醛灌注固定 ,取脑组织冰冻切片 ,用抗 GFAP 多克隆抗体孵育和 FITC 荧光标记二抗染色 ,再用抗 Brdu 单克 ( ) 隆抗体孵育和 Cy3 荧光标记二抗染色 ,在激光共聚焦显微镜下连续断层扫描 , 图像叠加 。结果 　清晰可见处于增殖期 S 期 细 胞胞核呈红色荧光 ,星形胶质细胞胞质呈绿色荧光 。结论 　影响免疫荧光双重标记样品效果的环节和因素很多 ,其中最重要 的因素是 2 个一抗的搭配和协调 。 [ 关键词] 　免疫荧光 ;双重标记 ;激光共聚焦显微镜 [ 中图分类号] 　R392. 33 　　　　[文献标识码] 　A Preparation of immunofluorescence double staining sample for laser confocal microscope LU Ju , SUN Wei , CHEN Deying ( Central Laboratory , College of High Altitude Military Medicine , Third Military Medical University , Chongqing 400038 , China) [ Abstract ] 　Objective 　To introduce the method of immunofluorescence double label for providing an ideal observation sample for laser scanning confocal microscope. Methods 　Rats were injected with Brdu intraperitoneally , and then fixed with perfusion of 40 gL paraformaldehyde. The brain tissue of the rat was frozen and sliced in the frozen microtome. The slices was separately stained with polyclonal antibodies of antiGFAP and monoclonal antibodies of antiBrdu , and then , incubated with IgG labeled by FITC and Cy3 individually. A continuous layer scanning of the slices and image overlapping were performed under laser confocal microscope.Results 　Red fluorescence was dyed in the nucleus at the proliferative phase ( S phase) and green fluorescence was shown in the cytoplasm of the astrocyte. Conclusion 　There are many factors which will influence the result of i