0.1 m cacodylate buffer

For ultrastructural analysis using Transmission electron microscopy (TEM), kidneys and podocyte cell cultures were fixed in 4% PFA and 2% glutaraldehyde (#4157, Carl Roth, Karlsruhe, Germany) in a 0.1 M cacodylate buffer (#11650, Science Services, München, Germany). The samples were postfixed in 1% osmium tetroxide (Science Services, #E19150) in double-distilled water (ddH₂O) and then washed six times in ddH₂O. The tissue was stained en bloc in 1% uranyl acetate solution (Science Services, #E22400-1) and washed two times in ddH₂O. Dehydration was performed via sequential incubation steps in 30%, 50%, 70%, 90%, and 2 × 100% ethanol (#32205, Fisher Scientific, Hampton, NH, USA) and then 2× 100% acetone (#179124, Sigma-Aldrich, St. Louis, MO, USA). All incubation steps were microwave-assisted (BioWave Pro+, PELCO, Fresno, CA, USA). After embedding in Durcupan resin (Sigma-Aldrich, #44611 and #44612), ultrathin sections (55 nm) were performed using a UC7 ultramicrotome (Leica), collected on Formvar-coated (Science Services, #E15830-25) copper grids (#G2500C, Plano GmbH, Wetzler, Germany). Post-staining was conducted for 1 min with 3% lead citrate (#11300, Delta Microscopies, Mauressac, France), and imaging was performed using a Talos L120c TEM (ThermoFisher, Waltham, MA, USA).

The intestine was fixed in 2% glutaraldehyde/2% formaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 48 h at 4 °C. Afterwards, samples were rinsed in 0.1 M cacodylate buffer (AppliChem) and post-fixed with 2% osmium tetroxid (Science Services) in 0.1 M cacodylate buffer for 2 h at 4 °C. Samples were dehydrated through an ascending ethanol series (AppliChem) and embedded in epoxy resin (Sigma-Aldrich). Ultrathin sections (70 nm) were cut with a diamond knife (Diatome) on an ultramicrotome (EM-UC6, Leica Microsystems) and placed on 100-mesh copper grids (Science Services). The sections were contrasted with 1.5% uranyl acetate (Plano) and lead citrate (Sigma-Aldrich). Images were acquired with a transmission electron microscope (JEM 2100 Plus, JEOL) and a OneView 4K camera (Gatan) with DigitalMicrograph software (v3.x, Gatan) at 80 kV at room temperature (RT). For each mouse and for each measured phenotype, ten random images were taken and the final score for each mouse was calculated as a mean value obtained from ten images. Imaging and scoring of electron microscope data were carried out under blinded conditions.