Hoechst fluka

Rat kidneys were snap-frozen and embedded in Tissue-Tek^® O.C.T. Compound (Sakura). Cryosections were fixed with 3.5% PFA, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) at RT for 15 min and blocked with CAS-block (Invitrogen). Sections were incubated with rabbit anti-CD2AP (Santa Cruz Biotechnology), quinea pig anti-nephrin IgGs (Sigma Aldrich) and goat anti-SHIP2 I-20 IgGs (Santa Cruz Biotechnology) overnight at +4 °C in ChemMate (Dako Cytomation, Glostrup, Denmark), followed by Alexa Fluor 555 donkey anti-rabbit, Alexa Fluor 488 donkey anti-quinea pig and Alexa Fluor 555 donkey anti-goat IgGs (Invitrogen) together with Hoechst (Fluka, Sigma Aldrich) for 1 h in ChemMate (Dako Cytomation). Samples were mounted in Mowiol and examined with Leica SP2 confocal microscope (Leica Microsystems CMS GmbH, Mannheim, Germany).

Differentiated podocytes were fixed with 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS and permeabilized with 0.1% Triton X-100 in PBS. Rat kidney cryosections were fixed with acetone. Samples were blocked with CAS-block (Invitrogen, Carlsbad, CA, USA), incubated with rabbit anti-PDK1 (Cell Signaling Technology, Danvers, MA, USA) and mouse anti-WT1 IgGs (Upstate, New York, NY, USA) for 1 h at room temperature (cells) or overnight at +4 °C (tissue sections) in ChemMate (Dako Cytomation, Glostrup, Denmark), followed by AlexaFluor 555 donkey anti-rabbit (Invitrogen) or DyLight 488 donkey anti-mouse IgGs (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA), and Hoechst (Fluka, Sigma-Aldrich) for 1 h in ChemMate (Dako Cytomation). Samples were mounted in Mowiol and examined with Zeiss Axiophot 2 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) or Leica SP2 confocal microscope (Leica Microsystems CMS GmbH, Mannheim, Germany).