Immunofluorescence Staining of TRPC1 and TRPV4 in Cells

The cells were fixed at 4% paraformaldehyde prepared in cytoskeletal buffer for 10 min at 37 °C [21 (link)] and washed with PBS 3 times. Then, the cells were blocked by bovine serum albumin for 1 h. Then, the primary anti-TRPC1 (1:1000; cat. no. #DF12783, Affinity Biosciences) and anti-TRPV4 (1:1000; cat. no. #DF8624, Affinity Biosciences) were incubated at 4 °C overnight, and the fluorescent secondary antibody Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (H + L) (1:500; cat. no. A0423; Beyotime Biotechnolog) was used to incubate at room temperature and away from light for 1 h. Finally, the 4',6-diamidino-2-phenylindole (DAPI, Sigma, USA) was used for re-staining the nucleus for 5 min and observed under THUNDER Imager Tissue (Leica, Germany).