Fluoro-Jade B染色与蛋白免疫荧光多标的染色方法

摘要

Objective To explore a method for combining Fluoro-Jade B (FJB) staining with immunofluorescent staining in rats with focal cortical infarction. Method Permanent distal middle cerebral artery occlusion (dMCAO) was induced in rats by electrocoagulation. The rat models were randomized into two groups, and frozen sections of the brain tissues from each group were stained with FJB followed by immunofluorescent staining or in the reverse order. Results FJB staining followed by immunofluorescence staining clearly visualized both FJB-positive and immunofluorescence-positive cells in the frozen sections, but the staining protocol in the reverse sequence failed to clearly show the immunofluorescence-positive cells. Conclusion FJB staining prior to immunofluorescence staining does not affect the staining effect of protein immunofluorescent staining and better visualizes the positive cells.%目的：寻找一种Fluoro-Jade B（FJB）染色结合目的蛋白免疫荧光多标的合适方法。方法通过电凝法制作大鼠大脑中动脉闭塞模型（MCAO），诱导皮层缺血半暗带神经元变性。实验分为FJB染色结合蛋白免疫荧光组以及目的蛋白免疫荧光结合FJB染色组。大鼠MCAO后5 d断头取脑，脑组织冰冻切片后，按上述两种染色顺序染色，观察两组实验双标的效果。结果 FJB染色结合目的蛋白免疫荧光组中，FJB和蛋白免疫荧光阳性细胞形态清楚，免疫双标效果良好；而蛋白免疫荧光结合FJB染色组，虽然FJB阳性细胞较为清晰，但与之免疫双标的蛋白免疫荧光阳性细胞不清楚，背景模糊，效果不佳。结论在两种免疫双标的方法中，先行FJB染色后再做目的蛋白免疫荧光染色，不会影响蛋白免疫荧光的染色效果，有利于后续实验的进行和观察。