Evaluation of the Immune Complex in Renal Biopsy of Dogs by Immunofluorescence of Formalin-Fixed and Paraffin-Embedded Section
Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
Renal biopsy is an essential tool for diagnosis of proteinuric kidney disease of dogs, and evaluation of the immune complex (IC) by immunofluorescence (IF) of frozen (F) sections is required for the diagnosis of IC-mediated glomerulonephritis (ICGN). However, the use of frozen sections is limited in diagnosis by renal biopsy because of certain limitations.
We aimed to develop a reliable IF method using formalin-fixed and paraffin-embedded (FFPE) sections to detect the IC in ICGN of dogs.
Renal biopsy specimens (n=12) were obtained from dogs with protein-losing nephropathies. FFPE sections were prepared, and eight antigen retrieval pretreatment methods were set as follows: non-treatment, digestion with trypsin (Try), microwave (MW) heating in citrate buffer (MW-CB; pH 6.0), MW in tris-EDTA buffer (MW-TE; pH 9.0), and combinations of the above. Then, the presence of IgG, IgA, IgM, and C3 were detected with Alexa Fluor 488-conjugated secondary antibodies.
Combination of Try (incubation for 30 min; Try-30) and MW-TE was the most effective for diagnosis, with clear positive signals being obtained for IgG, IgA, IgM, and C3 by IF-FFPE in all the samples. Granular signals, an important diagnostic indicator of ICGN, were clearly observed not only by IF-F but also by IF-FFPE after combined pretreatment with Try-30 and MW-TE. Signals for immunoglobulins and C3 by standard IF-F and the present IF-FFPE were almost completely matched in all samples.
The present IF-FFPE method can be used as an efficient tool in renal biopsy for diagnosis of proteinuric kidney disease of dogs.