微波石蜡切片免疫荧光多重标记技术在肾活检病理诊断中的应用

目的：肾活检组织免疫荧光检查常规是在冰冻切片上进行。本研究探讨了甲醛固定的肾活检组织进行石蜡切片免疫荧光多重染色在病理诊断中应用的可行性。方法：采用微波修复抗原法,对5例膜性肾病、5例狼疮性肾炎、5例IgA肾病和5例乙型肝炎病毒相关性肾炎患者肾组织石蜡切片分别进行IgG/Fn/Hoechst、C3/Fn/Hoechst、IgA/Fn/Hoechst、HBsAg/Fn/Hoechst免疫荧光多重染色。利用免疫荧光显微镜观察并照相。结果：微波修复抗原的石蜡切片进行免疫荧光多重染色,显示IgG、C3、IgA与HBsAg染为绿色,FN为红色,两者共表达的部位为橙黄色,细胞核为蓝色。石蜡切片的免疫荧光染色与用于病理诊断的冰冻切片免疫荧光染色结果一致。且石蜡切片较冰冻切片薄,结构清楚,免疫沉积物定位更准确,易判断结果。结论：微波石蜡切片免疫荧光多重染色,能在一张切片上同时显示两种以上抗原的共表达,为病理诊断提供更加直观详实的依据,可用于肾组织分子免疫病理检查,为使用石蜡切片进行回顾性免疫荧光诊断和研究提供了技术保障。

The Application of Microwave-assisted Multicolor Fluorescence Iimmunos-taining in Paraffin-embedded Renal Biopsy Tissues

Objective. to explore the practicality of multiple stainig with imrnunofluorescence in formalin- fixed and paraffin -embedded human renal tissue slices. Methods： Microwave technology permitted the development of new antigen labeling technique. The present study tested multicolor labeling of three antibodies in routinely processed paraffin- embedded renal biopsy tissue sections using a microwave- assisted immunofluorescence technique. Biopsy sarnples from patients with IgA nephropathy （ n = 5 ）, lupus nephritis （n= 5 ）, membranous nephropathy （ n = 5 ） and HBV related nephritis （ n = 5 ） were taken for simultaneous staining of IgG/Fn/Hoechst,C3/Fn/Hcechst, IgA/Fn/Hoechst and HBsAg/Fn/Hoechst. Antigen unmasking was performed with microwave treatment. The paraffin sections and frozen sections were analyzed by fluorescence microscope. Results： The multicolor fluorescence immunostaining of microwave - assisted paraffin sections showed that the stainings of IgG, C3, IgA and HBsAg were green, and FN was red. The coexpression of two different antigens was orange with blue nucleus. The results were in accordance with that of frozen sections. Furthermore, the paraffin sections were thinner and clearer, which lead to more specific location of immune deposits and easier judgment. Conclusion：The application of microwave- assisted immunofluorescence technique can show more than two antigens simultaneously in one paraffin section. This method is effective for revealing more accurate and direct details and useful for immune pathologic diagnosis of renal biopsy tissue.