Histofine reagent

Kidneys were immersed in 10% formalin and embedded in paraffin. Four-millimeter sections were processed for histopathology or immunohistochemistry. Glomerulosclerosis was defined by focal and segmental consolidation of the tuft by increased extracellular matrix, obliterating the glomerular capillary lumen. For the assessment of glomerulosclerosis, an examiner blinded to the experimental conditions assessed the percentage of glomeruli showing glomerulosclerosis with at least 50 glomeruli examined per field. For immunohistochemistry, paraffin embedded sections were stained with the following primary antibodies: rabbit anti-CD3 (DAKO, A 0452, 1:200), rat anti-F4/80 (AbDSerotec, MCA497, 1:500). CD3 and F4/80 staining were exposed with Histofine reagent (Nichirei Biosciences, 414141F) and color formation stopped by washing in phosphate-buffered saline. Slides were then counterstained with hematoxylin. Photomicrographs were taken with an Axiophot Zeiss photomicroscope (Jena, Germany). Quantification of CD3 and F480 staining was performed using ImageJ software (National Institutes of Health, Bethesda, MD).