不同固定与渗透方法对鸡胚骨骼肌卫星细胞免疫荧光显色的影响

Effect of different fixatives and permeabilization on immunofluorescence staining of skeletal muscle satellite cells in chick embryo

目的：探索有效的固定及渗透方法，以提高鸡胚骨骼肌卫星细胞免疫荧光显色的效果。方法：培养18胚龄鸡胚胸大肌卫星细胞，免疫荧光显色前采用4种不同的固定和渗透方法处理。95％乙醇固定15min95％乙醇固定，15min，再用0．25％TritonX-100穿透10min；先用4％多聚甲醛固定40min，再用0．25％TritonX-100穿透10min；预冷的丙酮固定10min。结果：乙醇＋TritonX-100处理的卫星细胞形态完整，细胞骨架蛋白结蛋白（Desmin）、生长分化因子-8（GDF-8）2种胞质蛋白的免疫荧光显色效果最好。结论：染色前采用95％乙醇固定15min结合0．25％TritonX-100穿透10min对鸡胚骨骼肌卫星细胞的免疫荧光显色效果更好。

Objective.. To explore efficient fixed and penetrative methods and improve the effectiveness of immunofluorescence staining of skeletal muscle satellite cells. Methods. Satellite cells of the major pectoral muscle at 18 embryo days were trea- ted by four different fixed and penetrative methods before immunofluorescence staining. Group A.. Satellite cells were fixed by 95% alcohol for 15 min~ Group B： Satellite cells were fixed by 95% alcohol for 15 rain, then permeabilizing by 0. 25% Triton X-100 for 10 min~ Group C.. Satellite cells were fixed by 4% paraformaldehyde for 15 min, then permeabilizing by 0. 25% Triton X-100 for 10 rain; Group D. Satellite cells were fixed by cooled acetone for 10 rain. Results. After detecting the expressions of desmin and GDF-8 which were expressed in cytoplasm, we concluded that group B was suit for immuno- fluorescence of skeletal muscle satellite cells of broilers. Conclusion： The method that satellite cells were fixed by 95 % alco- hol for 15 rain, then permeabilizing by 0. 25% Triton X-100 for 10 rain can not only keep antigens well, but also maintain morphological characteristics of skeletal muscle satellite cells, and is better for immunofluorescent staining of skeletal muscle satellite cells in chick embryos.