Dylight 488 donkey anti mouse iggs

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).

Differentiated human podocytes were fixed with 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS and permeabilized with 0.1% Triton X-100 in PBS. Podocytes were blocked with CAS-block (Invitrogen), stained with mouse anti-CDK2 (Santa Cruz Biotechnology) for one hour at RT in ChemMate (Dako Cytomation, Glostrup, Denmark), washed with PBS and incubated with DyLight 488 donkey anti-mouse IgG (Abcam) for one hour in ChemMate (Dako Cytomation). Perfused mouse kidney samples were fixed with 10% formaldehyde and embedded in paraffin. Deparaffinized sections were blocked with CAS-block (Invitrogen), incubated with rabbit anti-CDK2 (Abcam), guinea pig anti-nephrin (Progen Biotechnik GmbH, Heidelberg, Germany) or mouse anti-WT1 (Upstate, New York, USA) IgGs overnight at +4 °C in ChemMate (Dako Cytomation), washed with PBS and incubated with AlexaFluor 555 donkey anti-rabbit (Invitrogen), DyLight 488 donkey anti-quinea pig IgGs (Jackson Immuno Research Laboratories Inc., West Grove, PA) or DyLight 488 donkey anti-mouse IgGs (Jackson Immuno Research Laboratories Inc.). Samples were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined with Leica TCS CARS SP8 confocal microscope (Leica Microsystems CMS GmbH, Mannheim, Germany).