F4 80

3μm sections from kidneys fixed in paraformaldehyde stained. For antigen retrieval, sections were either incubated with 0.1% trypsin (Sigma Aldrich), 0.8% collagenase or microwaved in citrate buffer (pH 6.0). Primary antibodies were incubated overnight at 4°C by adding rabbit anti-mouse collagen IV antibody (1:400, Millipore, Darmstadt, Germany), rat anti-mouse CD31 (1:50, Dianova GmbH, Hamburg, Germany), rabbit anti-mouse active β-catenin (1:600, Cell signaling, Danvers, MA, USA), goat anti-mouse nephrin (1:500, R&D), rabbit anti-mouse WT-1 (1:200, Novus), rabbit anti-mouse collagen I (1:500 dilution, Abcam), rabbit anti-mouse CD45 (1:400, Abcam) and rat anti-mouse F4/80 (1:600, Biorad, Hercules, CA, USA). Slides treated with nonspecific antisera instead of primary antibody were used as negative control.
Collagen I, CD31, active β-catenin, F4/80, nephrin and collagen IV stained slides were scanned using the Aperio CSO system (Leica Biosystems, Buffalo Grove, IL, USA). The positive area was analyzed using Aperio Imagescope software. WT1-positive nuclei per glomerulus and CD45 positive cells per high power field were counted.

Mouse colons were fixed in neutral buffered formalin and embedded in paraffin. Sections at 3 μm were prepared and stained with H&E. IHC for CD3, B220 and F4/80 to identify T cells, B cells and macrophages, respectively, was performed on LeicaBiosystems Bond Autostainer. Sections were blocked with 2% normal rabbit serum (Vector Laboratories) for 20 minutes and subsequently incubated for 30 minutes with anti-CD3 (AbDSerotec), anti-B220 (BD Biosciences) or anti-F4/80 (eBiosciences) Ab. Biotinylated secondary antibody (anti-rabbit IgG), diluted 1:100 in 1.5% normal rabbit serum, was applied to the slides for 30 minutes and subsequent detection was achieved with Leica Biosystems’ Intense R detection Kit. IHC for BrdU was performed manually using anti-BrdU antibody (Invitrogen) and the Vectastain Elite Standard ABC kit (Vector Laboratories).