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本实验用间接免疫荧光法和电镜免疫胶金法显示培养细胞的胞内凝血Ⅷ因子相关抗原(ⅧR:Ag)反应阳性以及电镜下Weible-Palade(WP)小体存在,证明所分离并经多次传代的细胞为内皮细胞。材料和方法:1.从新生小牛肺动脉中以胶原酶消化法分离出内皮细胞,于199培养液中培养并传代,取第3-5代细胞。实验前细胞培养于涂有鼠尾胶原的载玻片上。2.WP小体检测:细胞以PBS(0.1M,pH=7.4)原位漂洗并分别以3％戊二醛45分钟、1％锇酸30分钟固定;将预聚好的Epon812块扣于经脱水和Epon812浸透的细胞单层上,聚合后用热烤法将细胞层剥离于载玻片,常规超薄切片和铅铀染色,透射电镜下观察。3.ⅧR:Ag检测:(1)间接免疫荧光法:以95％4℃乙醇原位固定细胞10分钟,
In this study, indirect immunofluorescence and electron microscopy immunogold method showed that the intracellular coagulation factor Ⅷ-related antigen (Ⅷ R: Ag) was positive in cultured cells and the presence of Weible-Palade (WP) bodies was observed under electron microscopy. Subcultured cells are endothelial cells. MATERIALS AND METHODS: 1. Endothelial cells were isolated from neonatal calf pulmonary arteries by collagenase digestion, cultured and subcultured in culture medium 199 and passaged 3-5 passages. The cells were cultured on rat microglia coated slides before the experiment. 2. WP body detection: Cells were rinsed in situ with PBS (0.1 M, pH = 7.4) and fixed with 3% glutaraldehyde for 45 min and 1% osmium tetroxide for 30 min respectively; Dehydration and Epon812 soaked cell monolayer. After polymerization, the cell layer was peeled off on a glass slide by hot-baking method, and conventional ultra-thin sections and lead uranium staining were observed under a transmission electron microscope. 3. Ⅷ R: Ag detection: (1) Indirect immunofluorescence: cells were fixed in situ with 95% ethanol 4 ° C for 10 minutes,