Dako fluorescent medium

Tissue was processed “free-floating” for immunofluorescence as follows. Briefly, sections were incubated with citrate buffer (0.01M, pH 6.0) for 1 min at 100°C. After this, sections were treated for 1 h with 5% normal donkey serum (NDS) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in PBS with 0.2% Triton- X100 (Sigma-Aldrich, St Louis, MO, USA) and were incubated overnight at room temperature in polyclonal rabbit IgG anti-cleaved caspase-3 (1:500, Cell Signaling, 9661) antibody. After washing, sections were incubated for 2 h with donkey anti-rabbit IgG conjugated with DL549 (1:200, Jackson Immunoresearch) for 2 h. After washing, sections were mounted on slides using Dako fluorescent medium (Dako North America, California). The sections were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) (dilution 1/500) (Sigma-Aldrich, St Louis, MO, USA) to identify cellular nuclei. The analysis of sections was performed using a confocal microscope (Leica TSC-SPE) using 40X objective. Serial stacks of the different regions (granule layer of dentate gyrus, RMS and olfactory bulb) were analyzed using ImageJ software. Interval between z planes was 1.15 μm.

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).