Podocyte Structural Dynamics in MWF Rats

Sample processing and subsequent imaging was performed as described before (Artelt et al., 2018 (link)). In short, 4 µm paraffin sections were directly mounted on coverslips (VWR). To correct for paraformaldehyde-induced autofluorescence, samples were incubated with 100 mM glycine in PBS for 10 min. Samples were blocked with 1% (v/v) fetal bovine serum, 1% (v/v) goat serum, 1% (v/v) bovine albumin and 0.1% (v/v) cold fish gelatin in PBS at room temperature for 1 h. Primary antibodies against nephrin (guinea pig, Progen GmbH, 1:100) and SSeCKS (Table S1; 1:200) were diluted in blocking solution and detected by a secondary anti-guinea pig antibody (1:800) and anti Cy3-labeled polyclonal goat anti-rabbit IgG (H+L) (1:600) (both from Jackson ImmunoResearch, Hamburg, Germany) diluted in blocking solution. Three-dimensional structured illumination microscopy (SIM) images were acquired using a Zeiss Elyra PS.1 system. Using Zeiss ZEN black software, 3D SIM images were reconstructed. Podocyte PEMP was performed using FIJI and a custom-build macro (Siegerist et al., 2017 (link)). Analysis was performed in two different MWF rat glomeruli. FSD was measured in eight (four sclerotic and four non-sclerotic) glomeruli.