Cy2 and cy3 conjugated secondary antibodies

For paraffin sections, samples were dehydrated and embedded in paraffin by standard procedures. Paraffin sections (2 µm) were cut on a Leica SM 2000R (Leica Microsystems, Wetzlar, Germany). After rehydration, sections were unmasked in citrate buffer (0.1 M, pH 6.0) by heating for 5 min in a pressure cooker. The nuclei were stained with 1 mg/100 ml Hoechst 33342 (Sigma-Aldrich) for 30 min. For immunofluorescence double-staining, samples were incubated with an antibody against zyxin (HPA004835 for human and HPA073497 for mice FFPE material; both from Sigma-Aldrich; IF dilution 1:100) and synaptopodin (61094, Progen Biotechnik GmbH, Heidelberg, Germany; IF dilution 1:100) overnight. Samples were washed with 1x PBS for 3 × 5 min and incubated with Cy2- and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; IF dilution 1:300) for 1 h. After additional washing, the samples were mounted in Mowiol (Carl Roth) for fluorescence microscopy. PAS stainings were performed by standard procedures. For transmission electron microscopy, kidneys were embedded in EPON 812 (SERVA, Heidelberg, Germany). Ultrathin sections were cut and contrasted with 5% uranyl acetate and lead citrate. All grids were examined with a LIBRA^® 120 transmission electron microscope (Carl Zeiss Microscopy, Jena, Germany). Scanning electron microscopy was performed according to Artelt et al. ^{80 (link)}

Antibody to LPL (SC-16657; Goat) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to GAPDH and TNF-α receptor 1 (TNFR1) were purchased from R & D Systems (Minneapolis, MN). Protein estimation reagent, molecular weight standards, and PAGE reagents were bought from Bio-Rad (Hercules, CA). Cy2- and Cy3-conjugated secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA). HRP-conjugated secondary antibodies for immunoblotting and the phosphoserine (p-Serine) antibody were purchased from Abcam (Cambridge, MA). Mounting solutions for mounting of coverslips were purchased from Thomas Scientific (Swedesboro, NJ) or Vector Labs (Burlingame, CA). Rhodamine-phalloidin and all other chemicals were purchased from Sigma (St. Louis, MO).

Antibody to L-plastin (SC-16657; Goat) was bought from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to GAPDH was purchased from R & D Systems (Minneapolis, MN) and Sigma (St. Louis, MO). Protein estimation reagent, molecular weight standards for proteins, and PAGE reagents were bought from Bio-Rad. Cy2- and Cy3-conjugated secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA). HRP-conjugated secondary antibodies for immunoblotting were obtained from GE Healthcare. Antibody to phosphoserine (p-Serine) was bought from Zymed laboratories (61–8100) or Millipore (AB1603). Alizarin red solution was bought from Life-line Cell Technology (CM-0058; Fredrick, MD) Rhodamine-phalloidin and other chemicals were purchased from Sigma (St. Louis, MO).