Immunohistochemical diagnosis of Alport's syndrome in paraffin-embedded renal sections: antigen retrieval with autoclave heating.
Alport's syndrome (AS) is a hereditary renal disease caused by mutations in the genes encoding collagen type IV. Immunohistochemical analysis of the alpha chains of collagen type IV has been found to be useful for the diagnosis of this disease. The monoclonal antibodies (mAbs) generated by us recognize alpha 1(IV) through alpha 6(IV) chains of collagen type IV on fresh-frozen sections but not on paraffin-embedded sections. Antigen retrieval by autoclave heating has been found to restore the epitopes recognized by the mAbs; however the heating conditions had not been well established. In this study, the heating conditions were carefully examined using renal sections obtained from AS and non-AS patients. The heating was performed in an autoclave, at 105 degrees -127 degrees C for 6-8 min. During the heating, the sections were immersed in 0.2 N HCl solution (pH 0.9). Then, the mAbs were applied for 30 min, and the bound mAbs were detected using the LSAB kit. The optimal temperature for the antigen retrieval varied among specimens, and was dependent on the type of basement membrane examined. Thus, it was considered that heating at two or three different temperatures could be helpful for the precise diagnosis of AS. Adopting the antigen retrieval method could extend the possibility of immunohistochemical diagnosis of AS to cases without using fresh-frozen sections.
Naito
Ichiro
I
Division of Ultrastructural Biology, Shigei Medical Research Institute, 2117 Yamada, Okayama 701-0202, Japan. inaito@shigei.or.jp
Ninomiya
Yoshifumi
Y
Nomura
Shinsuke
S
D000368:Aged; D000911:Antibodies, Monoclonal; D016022:Case-Control Studies; D002648:Child; D024141:Collagen Type IV / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005260:Female; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D008297:Male; D016253:Microscopy, Immunoelectron; D008875:Middle Aged; D009394:Nephritis, Hereditary / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016612:Paraffin Embedding
D000911:Antibodies, Monoclonal; D024141:Collagen Type IV