Sanchez A, Bilinski M, Gonzalez Nicolini V, Villar M J, Tramezzani J H
Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Argentina.
Histochem J. 1997 Aug;29(8):631-8. doi: 10.1023/a:1026492429668.
Cultured magnocellular neurons, isolated from adult rat supraoptic nuclei, were characterized by immunocytochemistry, using the avidin-biotin-peroxidase complex and antisera to vasopressin, oxytocin, galanin and cholecystokinin. Light microscope examination of the immunostained cultures revealed the presence of vasopressin- and oxytocin-like immunoreactivity, as well as neurons containing either galanin- or cholecystokinin-like immunoreactivity. In contrast, no significant galanin- or cholecystokinin-like immunoreactivity could be observed in freshly dispersed cells. Correlative scanning electron microscopical observations in the secondary electron imaging mode revealed that the stained neurons appeared significantly brighter than the unstained structures. Complementary observations with toad brain sections (preoptic area), immunostained for galanin, led to the same result. Considering previous results, it is suggested that the presence of galanin- and cholecystokinin-like immunoreactivity in the cultured neurons and its virtual absence in freshly dispersed cells is indicating a participation of these peptides in the regenerative processes taking place during culture. It is further concluded that the avidin-biotin-peroxidase method is suitable for correlative light and scanning electron microscopical studies of smooth surfaces and cultured cells.
从成年大鼠视上核分离出的培养大细胞神经元，通过免疫细胞化学方法进行鉴定，采用抗生物素蛋白-生物素-过氧化物酶复合物以及抗血管加压素、催产素、甘丙肽和胆囊收缩素的抗血清。对免疫染色培养物进行光学显微镜检查发现存在血管加压素样和催产素样免疫反应性，以及含有甘丙肽样或胆囊收缩素样免疫反应性的神经元。相比之下，在新鲜分散的细胞中未观察到明显的甘丙肽样或胆囊收缩素样免疫反应性。在二次电子成像模式下进行的相关扫描电子显微镜观察显示，染色的神经元比未染色的结构明显更亮。对蟾蜍脑切片（视前区）进行甘丙肽免疫染色的补充观察也得到了相同的结果。考虑到先前的结果，提示培养神经元中存在甘丙肽样和胆囊收缩素样免疫反应性，而在新鲜分散的细胞中几乎不存在，这表明这些肽参与了培养过程中发生的再生过程。进一步得出结论，抗生物素蛋白-生物素-过氧化物酶方法适用于对光滑表面和培养细胞进行相关光学显微镜和扫描电子显微镜研究。
