Paraffin immunofluorescence for detection of immune complexes in renal biopsies: an efficient salvage technique for diagnosis of glomerulonephritis in dogs.

BACKGROUND: Renal biopsy is an essential tool for the diagnosis of proteinuric kidney diseases in dogs, and evaluation of immune complexes (IC) by immunofluorescence (IF) of frozen sections (IF-F) is required for the diagnosis of IC-mediated glomerulonephritis (ICGN). However, the use of frozen sections from renal biopsies can have limitations. The aim of this study was to develop a reliable IF method using formalin-fixed and paraffin-embedded (FFPE) sections to detect ICs in dog ICGN.

METHODS: Renal biopsy specimens were obtained from dogs with protein-losing nephropathies. FFPE sections were prepared, and eight antigen retrieval pretreatment protocols were performed: digestion with trypsin, microwave (MW) heating in citrate buffer (MW-CB; pH 6.0), MW heating in Tris-EDTA buffer (MW-TEB; pH 9.0), as well as combinations of the above, and a non-treated control.

RESULTS: A combination of trypsin for 30 min (Try-30) and MW-TEB; pH 9.0 was the most effective antigen retrieval pretreatment, with clear positive signals for IgG, IgA, IgM, and C3 detected by IF-FFPE. Granular signals, an important diagnostic indicator of ICGN, were clearly observed by both IF-F and IF-FFPE after combined pretreatment with Try-30 and MW-TEB, and IgG, IgA, IgM, and C3 signals were almost completely matched in all samples by IF-F and IF-FFPE.

CONCLUSION: IF-FFPE with Try-30 and MW-TEB pretreatment is a valuable technique for the diagnosis of renal diseases in dogs. This method could be an efficient tool when standard IF-F cannot be used, or does not provide useful results due to lack of glomeruli in the specimens for IF-F.