冷冻超薄切片免疫电镜技术在肾活检病理中的应用

Ultrathin cryo-sections immunoelectron microscopy technique in the application of renal pathology

目的:介绍冷冻超薄切片免疫电镜技术在肾活检病理中的应用。方法:肾活检组织经固定、蔗糖冷冻保护后,应用改良Tokuyasu技术冷冻超薄切片进行IgA、κ、λ、C3aR、Podocine、Nephrin免疫胶体金标记,结合免疫荧光标记,并与Epon812常规免疫电镜技术及LR White Resin低温包埋免疫电镜技术进行对比,观察和比较冷冻超薄切片免疫电镜方法对肾活检组织超微结构、抗原保存及免疫标记的影响。结果:冷冻超薄切片肾组织免疫电镜微细结构保存良好,细胞内结构清晰,肾组织及细胞内抗原保存较好,但细胞内基质、糖原保存不理想。IgA、κ、λ免疫标记金颗粒密度高,分布特异、敏感性高。Epon812常规免疫电镜和LR White Resin低温包埋免疫电镜方法能很好地标记C3aR、Podocine、Nephrin较难检测的抗原。结论:肾活检冷冻超薄切片免疫电镜方法免疫标记特异度强、敏感度高,是研究肾脏病理超微结构较为理想的方法。

Objective： To investigate the application of ultrathin cryo-immunogold electron microscopy methods in renal biopsy. Methodology： Tissue blocks of renal biopsy were fixed in fixation solution, and further sucrose cryoprotected. Ultrathin cryosections were cut with the application of improved technology of Tokuyasu. The grids were immunolabelled using IgA, ？, ？, C3aR, podocine, and nephrin. Combined with immunofluorescence markers, immunoelectron microscopy for routine Epon812 and LR White embedding immunoelectron microscopy at low temperature as a reference, ultrathin cryosections were observed and the affects of those methods on the ultrastructure of renal biopsy, antigen preservation and immunolabelling were compared. Results： The ultrathin cryosections of renal tissue well preserved, fine structure. The intracellular structure was also clear while intracellular antigens preservation was good. However, the intracellular matrix, stored glycogen was not ideal. IgA 、？、？ immune markers of gold particles density was high, distribution specificity and high sensitivity. The antigens of C3aR, podocine and nephrin , which might be difficult to detected by Epon812 routine immune electron microscopy and LR White embedding immunoelectron microscopy, were well marked, and the immune markers of gold particle showed high density and distribution of specific and sensitive. Conclusion： The immunogold labeling of renal biopsy ultrathin cryosections was specific and high sensitive. This technique was a more satisfactory method at the ultrastructure level of renal morphological changes.