免疫荧光双重染色的激光共聚焦显微镜样品制备及观察

Preparation of immunofluorescence double staining sample for laser confocal microscope

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 g/L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。

Objective To introduce the method of immunofluorescence double label for providing an ideal observation sample for laser scanning eonfocal microscope. Methods Rats were injected with Brdu mtraperitoneally, and then fixed with perfusion of 40 g/L paraformaldehyde. The brain tissue of the rat was frozen and sliced in the frozen microtome. The slices was separately stained with polyclonal antibodies of anti-GFAP and monoclonal antibodies of anti-Brdu, 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 immunofluorescence double staining. One of the most important factors is the arrangement and coordination in the pair of the first antibodies.