Goat anti nephrin

Immunofluorescent microscopy was performed as discussed in our previous publications [23 (link), 24 (link)]. Briefly, the kidneys were perfused in situ and then fixed with fresh 4% PFA and stored at −80°C. Subsequently, paraffin sections (4 µm) were prepared and de-paraffinized in xylene and re-hydrated through graded concentrations of alcohol. Epitope retrieval was carried out by heating the samples at 98 °C for 2 h in Retrieveall-1 (Sign et Laboratories, Inc.). Subsequently, cooled samples were permeabilized with 0.3% Triton X-100 for 10 min, and were blocked with 2% BSA in 0.1% Triton X-100 for 1h at room temperature. Sections were then incubated with primary antibodies overnight at 4°C, followed by Alexa Fluo r secondary antibodies (Invitrogen, 1:800), donkey anti-rabbit IgG Alexa Fluor 568 or donkey anti-goat lgG Alexa Fluor 488, for 1 hour at room temperature. Primary antibodies included rabbit anti-Grem2 (Novus Biologicals, NBP1–31150, 1:100), goat anti-nephrin (R&D system, 1:100). All antibodies were diluted in 0.1% Triton X-100, 2% BSA in PBS. Cells were then counterstained with Hoechst33342 (Sigma-Aldrich) to identify nuclei. Morphological changes were visualized and captured with a ZEISS microscope (Carl Zeiss MicoImaging GmbH, Jena, Germany) equipped with a digital imaging system.