Cy3 or cy2 conjugated donkey anti mouse or donkey anti rabbit igg

Kidney cryosections were fixed with 3.7% paraformalin for 15 min at room temperature. HK-2 or NRK-49F cells cultured on coverslips were fixed with cold methanol: acetone (1:1) for 15 min at room temperature, followed by blocking with 0.3% Triton X-100 (Sigma-Aldrich) for 15 min and 10% normal donkey serum in PBS for 1 h at room temperature. Slides were then incubated with primary antibodies at 4 °C overnight. After washing, the slides were then incubated with Cy3 or Cy2-conjugated donkey anti-mouse or donkey anti-rabbit IgG (Jackson Immuno-Research Laboratories, West Grove, PA) at room temperature for 2 h. Nuclei were stained with DAPI (Sigma-Aldrich) according to manufacturer’s instruction. The slides were then observed under a confocal microscope (Leica SP8; Leica Microsystems, Buffalo Grove, IL).

Frozen kidney sections (3 µm) were fixed with 4% paraformalin for 15 minutes at room temperature. HKC‐8 cells cultured on coverslips were fixed with cold methanol:acetone (1:1) for 10 minutes at −20°C. The slides were blocked with normal donkey serum and incubated with primary antibodies as follows: anti‐fibronectin (F3648, Sigma‐Aldrich), anti‐β‐catenin antibody (ab15180; Abcam), anti‐STAT3 antibody (4904; Cell Signaling Technology), anti‐STAT6 antibody (5397; Cell Signaling Technology) and anti‐E‐cadherin (3195; Cell Signaling Technology). After washing, the slides were incubated with Cy3‐ or Cy2‐conjugated donkey anti‐mouse or donkey anti‐rabbit IgG (Jackson Immuno‐Research Laboratories, West Grove, PA). Nuclei were stained with DAPI (Sigma‐Aldrich) according to the manufacturer's instruction. Images were taken by confocal microscopy (Leica TCS SP2 AOBS; Leica Microsystems, Buffalo Grove, IL) or Olympus DP80 microscope with EMCCD camera.