Ab15180

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).

For immunohistochemical staining, 3‐μM‐thick paraffin sections of kidney samples were prepared. Periodic acid‐Schiff (PAS) and Sirius red staining were performed by a standard protocol or according to the manufacturer's instructions. Immunohistochemical staining was performed using routine protocol as described previously.
²⁴ (link) Images were taken with an Olympus DP80 microscope with an EMCCD camera. Primary antibodies used were as follows: anti‐CXCR4 (PA1237; Boster), anti‐fibronectin (F3648; Sigma‐Aldrich), anti‐α‐SMA (A2547; Sigma‐Aldrich), anti‐γH2AX (A11463; ABclonal), anti‐P16^INK4A (sc‐1661; Santa) and anti‐β‐catenin (ab15180; abcam).

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.

Kidney sections and immunohistochemical staining were performed as previously described (Liu et al., 2020 (link)). Primary antibodies included rabbit polyclonal anti–Wnt1 (ab15251; Abcam, Inc.), rabbit polyclonal anti-β-catenin (ab15180; Abcam, Inc.), and rabbit polyclonal anti-fibronectin (F3648; Sigma-Aldrich).

Immunohistochemical staining was performed using the established protocol (He et al., 2012 (link)). Antibodies used were as follows: rabbit anti-fibronectin (F3648; Sigma), mouse anti-α-SMA (ab7817; Abcam), mouse anti-β-catenin (610154, BD Biosciences) and rabbit anti-β-catenin (ab15180, Abcam). After incubation with the primary antibodies at 4°C overnight, slides were then stained with Biotin-SP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). Images were captured by using OLYMPUS BX43 microscope equipped with a digital camera.