Anti keratin

HDPFs at the third passage were fixed with 4 % paraformaldehyde (PFA) for 30 min at room temperature and then incubated in PBS containing 0.4 % Triton X-100 for 10 min on ice and then blocked with 2 % bovine serum albumin (BSA) for 60 min at 37 °C.
After the blocking step, the cells were incubated with primary antibody-anti-vimentin (1:100, Boster, Wuhan, China), anti-keratin (1:100, Boster) at 4 °C overnight; PBS was used as the negative control. The cells were then washed with PBS and incubated for 1 h with the secondary antibodies, namely anti-mouse IgG Alexa Fluor-488 or at antirabbit IgG Alexa Fluor-594 1:1000 at room temperature (Life Technologies, Paisley, UK). Glass cover slips were mounted using mounting media supplemented with DAPI stain (VectorLabs, Peterborough, UK) and preparations imaged under a fluorescent microscope (Olympus, Tokyo, Japan).

Podocytes in six-well plates were xed with 4% paraformaldehyde at room temperature for 30 min, incubated in phosphate-buffered saline (PBS) containing 0.4% Triton X-100 for 10 min, and blocked with 2% bovine serum albumin at 37°C for 60 min. The cells were then incubated with the primary antibodies anti-vimentin (1:100; Boster Biological Technology, Wuhan, China) and anti-keratin (1:100; Boster Biological Technology) at 4°C overnight. PBS was used as the negative control. Then, the cells were washed with PBS and incubated with anti-mouse IgG Alexa Fluor 488 or anti-rabbit IgG Alexa Fluor 594 (1:1000; Life Technologies, Paisley, UK) for 1 h at room temperature. The glass cover slides were installed using the installation medium with 4 ,6-diamidino-2-phenylindole dye (Vector Labs, Peterborough, UK) and prepared for imaging under a uorescence microscope under ⊆200 magni cation (Olympus, Tokyo, Japan).