Cy2 or cy3 conjugated donkey anti mouse or anti rabbit igg

Kidney cryosections were fixed with 4% paraformaldehyde solution for 15 min at room temperature. Primary antibodies were as follows: anti‐active‐β‐catenin (4270s; Cell Signaling Technology), anti‐TOMM20 (ab186735; Abcam), anti‐Lotus Tetragonolobus Lectin (LTL) (FL‐1321; VECTOR Laboratories), anti‐Peanut Agglutinin (PNA)(FL‐1071; VECTOR Laboratories), anti‐Dolichos Biflorus Agglutinin (DBA)(FL1031; VECTOR Laboratories). After washing with TBS‐T, slides were incubated with Cy2 or Cy3‐conjugated donkey anti‐mouse or anti‐rabbit IgG (Jackson Immuno‐Research Laboratories, West Grove, PA). Nuclei were stained with DAPI (C1006, Beyotime) according to the manufacturer's instructions. Images were taken by confocal microscopy (Leica TCS SP2 AOBS, Leica Microsystems, Buffalo Grove, IL).

HK‐2 cells cultured on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by permeabilizing with 0.2% of Triton X‐100 (T8787; Sigma‐Aldrich) for 10 min and blocking with 10% of donkey serum for 1 h. Paraffin‐embedded mouse kidney sections (3 μm thickness) were prepared by a routine procedure. Then the slides were immunostained with special primary antibodies overnight at 4°C. The primary antibodies included: anti‐fibronectin (F3648; Sigma‐Aldrich), anti‐Flag (M185‐3S, MBL), anti‐ADRP (ab52356; Abcam), β‐catenin (ab15180; abcam), anti‐CXCR4 (sc‐53,534; Santa), anti‐CPT1A (ab128568; abcam), anti‐Lotus Tetragonolobus Lectin (LTL) (FL‐1321; VECTOR Laboratories), anti‐Peanut Agglutinin (PNA) (FL‐1071; VECTOR Laboratories) and anti‐Aquaporin 3 (AQP3) (ab125219; abcam). After washing, slides were incubated with Cy2‐ or Cy3‐conjugated donkey anti‐mouse or anti‐rabbit IgG (Jackson Immuno‐Research Laboratories, West Grove, PA). Nuclei were stained with DAPI (C1006, Beyotime) according to the manufacturer's instructions. Images were taken by confocal microscopy (Leica TCS SP2 AOBS, Leica Microsystems, Buffalo Grove, IL).