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人脑胶质瘤细胞系BT_(325)细胞用含Triton X-100的缓冲液处理后,用针对波形纤维蛋白(vimentin)的单克隆抗体,与胶质纤维酸性蛋白(GFAP)抗血清进行间接免疫荧光染色。在荧光显微镜下观察后,将细胞用酒精逐级脱水,然后进行临界点干燥。将干燥后的细胞进行碳-铂喷镀,所得到的碳-铂复型用透射电镜观察。用本法可清晰地观察到完整的细胞骨架,以及微管、微丝与中间丝的形态与分布。在经vimentin单克隆抗体免疫染色的细胞内,中间丝直径较粗;在用GFAP抗血清染色后,只有部分细胞内中间丝直径较粗。如将Triten处理的细胞先用甲醛固定后,再进行免疫染色,中间丝直径增加较少;但如果先进行免疫染色后再用甲醛固定,中间丝直径显著增加。这与免疫荧光观察结果相一致,说明甲醛固定对中间丝分子上的抗原位点起破坏或遮蔽作用。
Human brain glioma cell line BT_ (325) cells were treated with Triton X-100-containing buffer and then directly linked to glial fibrillary acidic protein (GFAP) antiserum using a monoclonal antibody against vimentin Immunofluorescence staining. After observation under a fluorescence microscope, the cells were dehydrated stepwise with alcohol, and then the critical point was dried. The dried cells were subjected to carbon-platinum plating, and the obtained carbon-platinum double-type was observed with a transmission electron microscope. Using this method, the integrity of the cytoskeleton and the morphology and distribution of microtubules, microfilaments and intermediate filaments can be clearly observed. In the cells immunostained with the vimentin monoclonal antibody, the diameter of the intermediate filament was coarser; only some of the intracellular filament diameters were thicker after staining with GFAP antiserum. For example, Triten-treated cells were first fixed with formaldehyde and then immunostained, and the diameter of the intermediate filament increased less; however, if the cells were first fixed with formaldehyde after immunostaining, the diameter of the intermediate filament significantly increased. This is consistent with the results of immunofluorescence, indicating that formaldehyde fixation destroys or masks the antigenic sites on the intermediate filament molecules.