Rs23220

The cells were seeded in a 24-well plate with poly-L-lysine glass slides and cultured in an incubator for 24 h, after which the medium was removed, and the cells were washed with phosphate-buffered saline (PBS, No. P1010, Solarbio, Beijing, China). The cells were then sequentially treated with 4% paraformaldehyde (C11325432, Macklin, Shanghai, China), 0.5% Triton X-100 (MKBQ0896V, Sigma-Aldrich, St. Louis, MO, United States), and 5% BSA (A8020, Solarbio, Beijing, China). The cells were gently washed with PBS each time when the reagent was changed. The treated cells were incubated with β-actin (13E5) Rabbit mAb (4970, CST, MA, United States) overnight. The antibody was recovered, washed with PBS, and then incubated with goat anti-rabbit IgG (RS23220, Immunoway, Shanghai, China) mixed with DAPI (097M4085V, Sigma, St. Louis, MO, United States) for 2 h in the dark. After washing with PBS, the slides were removed, placed on the slides with anti-fluorescence attenuation mounting tablets (10142592, Dako, Copenhagen, Denmark), and observed under a fluorescence inverted microscope.

Immunofluorescence staining was performed on paraffin sections. The tissue sections were processed for the primary antibody incubation [31 (link)]. The primary antibodies against nephrin (1 : 500, Abcam, Cambridge, UK), WT1 (1 : 50, Abcam, Cambridge, UK), and AQP1 (1 : 200, Abcam, Cambridge, UK) were incubated at 37°C for 1 h, followed by the incubation with secondary antibodies (goat anti-rabbit lgG, RS23220, ImmunoWay, USA) for 1 h at 37°C. The sections were counterstained with DAPI (D9542, Sigma, USA) to visualize the nuclei and observed using a digital pathology scanner (VS120–S6–W, OLYMPUS).