Horseradish peroxidase labelled secondary antibody

Immunofluorescence and immunofluorescence staining was performed by the ways described.²² After incubated with the antibodies that against HDAC6 (1:100; Cloud‐Clone Corp; PAE906Mu01), synaptopodin (1:100; Santa Cruz; sc‐515842), LC3‐II (1:100; Abcam; ab48394) or ac‐α‐tubulin (1:100; Abcam; ab24610) at 4°C overnight, the washed frozen sections were incubated with secondary antibodies (1:200 dilution; Alexa Fluor, Life Technologies) for 1 hour in a darkroom before imaging by confocal microscopy (Radiance 2000; Bio‐Rad). Images were collected and then using PRISM software (API, USA) to process data, and finally, arranged into figures using Adobe Photoshop. The kidney sections were incubated with anti‐WT1 (1:50; Abcam; ab89901) and then with horseradish peroxidase‐labelled secondary antibody (Beyotime) for immunohistochemistry. WT1‐positive cells per glomerulus were counted by two renal pathologists in a blinded method.

Western blot was performed as previously described (Ahn et al. 2019 (link)). The membranes were incubated with the primary antibodies: rabbit anti-TGF-β1 (1:1000, Proteintech, USA), rabbit anti-α-smooth muscle actin (α-SMA) (1:2000, ImmunoWay Biotechnology, USA), rabbit anti-collagen I (1:2000, Abcam, UK), rabbit anti-Smad3 (1:1500, Abcam, UK), rabbit anti-p-Smad3 (1:1500, Abcam, UK) or rabbit anti-β-actin (1:1500, Beyotime Institute of Biotechnology, China) overnight at 4 °C. The membranes were processed with the horseradish peroxidase-labelled secondary antibody (1:2000, Beyotime Institute of Biotechnology, China). The bands were visualised using the ECL detection reagents (Beyotime Institute of Biotechnology, China). The ratio of the density of bands of the detected protein to that of β-actin was used for statistical analysis.

Cryosections with a thickness of 4 μm were prepared using a cryostat and were fixed in 4% paraformaldehyde for 15 min. After blocking, the cryosections were incubated with primary antibodies and then with a fluorescein Cy3-FITC-labelled secondary antibody (1:100; Proteintech, Wuhan, China). Fluorescence images were recorded using a TCS SP5II confocal microscope (Leica, Bensheim, Germany). The following primary antibodies were used: anti-desmin (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-podocalyxin (1:100; R&D Systems, Minneapolis, MN, USA), and anti-snail2 (1:100, Proteintech). Podocytes were seeded onto clean glass coverslips, fixed with 4% paraformaldehyde, and permeabilised with 0.2% Triton X-100. The slides were incubated with an anti-PCNA (1:100, Proteintech), anti-synaptopodin (1:100; Proteintech), anti-CDK4 (1:100; Abcam, Cambridge, UK), anti-P-YAP (1:100; Cell Signaling Technology, Danfoss, MA, USA), or anti-snail2 (1:100, Proteintech) antibody.
For immunohistochemistry analysis, after deparaffinisation, rehydration, antigen retrieval, and blocking, the sections were incubated with an anti-PCNA (1:100), anti-CDK4 (1:100), anti-desmin (1:100), anti-YAP (1:100, Proteintech), or anti-cyclin D1 (1:100, Cell Signaling Technology) primary antibody and then with a horseradish peroxidase-labelled secondary antibody (Beyotime, Shanghai, China).