锇酸固定组织的包埋后免疫电镜胶体金标记方法的改进

目的建立一种适用于中枢神经系统蛋白质抗原定位的包埋后免疫电镜胶体金标记方法。方法利用还原锇酸固定大鼠脑组织切片,首先在冰冻漂浮切片上用NaIO4确定待检抗原的可修复性,然后在亲水树脂Technovit7100包埋的半薄切片寻找使抗原得以最大程度修复的最佳NaIO4浓度,最后在超薄切片上进行相应抗原的免疫胶体金标记。结果NaIO4对不同抗原的修复作用明显不同。对于可修复抗原,低浓度NaIO4即可以使其充分修复。低浓度NaIO4处理组织可使组织超微结构保持完好,并可使胶体金高密度标记。

Improvement of a post-embedding immunogold labeling method for osmicated tissue

Objective To establish a new post-embedding immunoelectron microscopy method for localization of protein antigens in central nervous system using colloidal golds as probes.Methods Rat brain tissue sections were fixed with osmium-ferrocyanide mixture.Free floating frozen sections were collected to determine the retrievability of antigens by sodium metaperiodate(NaIO_(4)).Then the best concentration of NaIO_(4) for maximal antigen retrieval was determined on hydrophilic Technovit 7100 resin-embedded semithin sections.Finally,the corresponding antigen was labeled with colloidal golds on ultrathin sections.Results NaIO_(4) showed different retrieving effects on different antigens;to those antigens that could be effectively retrieved by NaIO_(4),low concentration of NaIO_(4)could realize antigen retrieval;EM microscopy showed that ultrathin sections treated by low concentration of NaIO_(4) allowed higher density of colloidal gold labeling without apparently compromising the ultrastructures of tissues.