Immunofluorescence Staining of Podocyte Proteins

Cryosections (6 μm) were cut on a Microm HM 560 microtome (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and immunofluorescence staining was performed as previously described [21 (link)]. Nephrin staining was carried out overnight at 4°C with 1:2000 rabbit anti-zebrafish nephrin (gift of Dr. A. Majumdar, Uppsala, Sweden) and Alexa-Fluor-647 anti-rabbit antibody (Invitrogen, Carlsbad, California, USA). An antigen retrieval step was necessary for podocin staining. Briefly, cryosections were boiled in Tris-HCL pH 9.0 for 10 min. After antigen retrieval and blocking, cryosections were incubated with a rabbit anti-podocin antibody (Proteintech, Rosemont, Illinois, USA; 1:200) o.n. at 4°C followed by an incubation with Alexa-Fluor-647-labeled anti-rabbit antibody (Invitrogen). Nuclei were stained with Hoechst 33342 (Sigma-Aldrich) and sections were finally mounted with Mowiol (Carl Roth, Karlsruhe, Germany). Confocal microscopy of cryosections was carried out on a Leica TCS SP5 with a 40x and a 63x oil immersion objective.