Mab1263

Each section was incubated overnight at 4 °C with primary antibodies targeting Type I procollagen (ab64409, rat mAb, Abcam, Cambridge, UK, cross-reacts with human), Type I collagen (600–401-103, rabbit pAb, Rockland, Limerick, PA, cross-reacts with bovine and human), Type III collagen (ab6310, FH-7A, mouse mAb, Abcam, Cambridge, UK, cross-reacts with rat and human), Type V collagen (AM10159PU-N, V13F6, mouse mAb, Acris, San Diego, CA, cross-reacts with human), keratin-14 (20R-CP002, guinea pig pAb, Fitzgerald Industries International, Acton, MA) and PDGFRbeta (MAB1263, mouse mAb, R&D Systems, Minneapolis, MN). Primary antibodies were detected using Alexa488- or Alexa594-conjugated secondary antibody (Thermo Fisher Scientific). Sections were examined with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and images were captured with a DP72 controller digital camera (Olympus). The staining intensity in 6 randomly selected microscopic fields was quantified by using WINROOF2015 image-analyzing software (Mitani, Fukui, Japan, https://www.mitani-visual.jp/products/image_analys_ismeasurement/winroof/), as described previously^{10 (link)}.

Human frozen kidney sections were fixed in ice-cold acetone and blocked using 5% normal goat serum solution for 1 h at room temperature. Mouse kidneys were fixed with 4% paraformaldehyde and subsequently embedded with paraffin. Paraffin-embedded kidney sections (4 μm) were processed with antibodies to the antigens described below. For immunofluorescent studies, after HIER treatment with Tris–EDTA pH 9 for 20 min at 100 °C, sections were blocked using 5% normal goat serum solution for 1 h at room temperature.
Frozen and/or paraffin sections were incubated with NPR3 (ab97389, abcam, 1:1000), Synaptopodin (61094; Progen, 1:700), CD31 (303105; Biolegend, 1:500), PDGFRβ (MAB1263; R&D System, 1:1000), Nephrin (BP5030; Origene, 1:200), Wilms-Tumor1 (WT1) (ab89901; abcam, 1:100), ⍺-smooth muscle actin (A5228; Merck, 1:1000), tdTomato (TA150128; Origene, 1:50) at 4 °C overnight. Alexa Fluor conjugated secondary antibodies (Invitrogen) were used for visualization. All slides were co-stained with Hoechst 33342 (H3570, Life Technologies: 1:10,000). For paraffin embedded sections an extra step of incubation with sudan black B (0.1% in 70% ethanol, #199664, Sigma-Aldrich) solution was applied before mounting in Dako fluorescent medium (S3023, Agilent).