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2006年1月13日Journal Reading By Jacky Lee INTRODUCTION Microwave treatment（MW）plus protease digestion or trypsin digestion（Try）to demonstrate the deposition of immunoglobulin（Ig）or complements in formaldehyde-fixed and paraffin-embedded（FFPE）renal biopsy tissue Routine procedure for diagnosis of renal biopsy Immunofluorescence（IF） frozen renal tissues diagnosis of glomerulonephritis Electron microscopy（EM） Hematoxylin and Eosin stain Histochemical stain fixation of tissues diagnosis and evaluation of the disease stage INTRODUCTION No glomerulus in frozen tissue → FFPE tissue Denatured Antigens → Antigens retrieval Trypsin digestion Microwave treatment Mixed method Focus comparison： Microwave treatment plus protease digestion Trypsin digestion Conventional immunofluorescence METHODS Renal Biopsy Samples（Shinshu University School of Medicine） （7）IgA nephritis （7）lupus nephritis （2）mesangial lupus nephritis （3）membranous lupus nephritis （2）diffuse lupus nephritis （7）membranous nephropathy （2）stage Ⅰ （2）stage Ⅱ （3）stage Ⅲ （3）mesangiocapillary glomerulonephritis（MPGN） METHODS Tissue Process Fixed in 30％ formaldehyde Paraffin embedded 3 μm section on silane coating slide Immunological analysis Direct immunofluorescence Indirect method Antigen unmasking methods Microwave treatment plus protease digestion Trypsin digestion METHODS Direct immunofluorescence Deparaffin （HemoD） Rehydrate（ethanol） Wash（PBS） Antigen unmasking Wash（PBS） FITC-labelled Rabbit antihuman IgA, IgG, IgM and C3 antibodies（DAKO）4℃ o/n Wash（PBS）… METHODS Indirect mothod Deparaffin （HemoD） Rehydrate（ethanol） Block endogenous peroxidase（1％ H2O2 in methanol） Antigen unmasking Rabbit antihuman IgA, IgG, C3c and C1q antibodies（DAKO, Kyoto, Japan）4℃ o/n Goat antirabbit Ig Ab conjugated with peroxidase（DAKO）37℃ 1hr DAB… METHODS Antigen unmasking Microwave treatment（MW-0） 0.1 mol EDTA pH 8.0 for 10 min Microwave treatment plus protease digestion MW ＋ 0.05％ protease （Sigma, type VII）in PBS for 10 min（MW-10）, 30