Poly bedr 812

Late stage embryos of the desired genotype were prepared for TEM analysis essentially as described [65 (link)]. Following washing with 0.1 M cacodylate buffer, tissue was stained with 1% osmium tetroxide for 1 hour, dehydrated in a graded ethanol series and propylene oxide and embedded in Poly/BedR 812 (Polysciences, Inc., Eppelheim, Germany). Semi-thin cross sections of each 1 μm were made on a Leica Ultracut S ultramicrotome. Sections were stained with toluidine blue for 3 minutes at 70°C. Image acquisition was on a Zeiss Axiophot microscope system. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined with a FEI Morgagni electron microscope. Digital images were taken with a Morada CCD camera and the iTEM software (Olympus Soft Imaging Solutions GmbH, Münster, Germany).

Tg(fabp10a:gc-EGFP) embryos at 120 hpf were fixed in 4% formaldehyde/0.5% glutaraldehyde (EM-grade) in 0.1 M phosphate buffer for 2 hr at RT. For knockdown analysis embryos were injected with tmem63c ex2-sgRNA of a concentration of 250 ng/µl to enhance the observed phenotype (Figure 7E,F). Prior to analysis embryos were sorted for a clear knockdown phenotype. Samples were stained with 1% OsO₄ for 2 hr, dehydrated in a graded ethanol series and propylene oxide and embedded in Poly/Bed^R 812 (Polysciences, Eppelheim, Germany). Ultrathin sections were contrasted with uranyl acetate and lead citrate. Sections were examined with a FEI Morgagni electron microscope and a Morada CCD camera (EMSIS GmbH, Münster, Germany). Image acquisition and quantification of podocyte foot process width and number of slit diaphragms per µm GBM was performed with the iTEM software (EMSIS GmbH, Münster, Germany).