Ab84818

Cells were fixed in 4% paraformaldehyde (Boster) for 30 min, and washed in PBS (10 min × 3 times). To block non-specific epitopes, cells were incubated with immunostaining blocking buffer (Beyotime Institute of Biotechnology) for 60 min. Then cells were incubated overnight at 4°C with primary antibodies: c-kit (sc-1494, 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HCN1 (ab84816, 1:100), HCN2 (ab65704, 1:100), HCN3 (ab84818, 1:100) and HCN4 (ab69054, 1:100) (all from Abcam), followed by the appropriate fluorescence-conjugated secondary antibodies: Alexa Fluor 488 mouse anti-goat IgG (bs-0294M, 1:200; Bioss, Beijing, China), Alexa Fluor 647 goat anti-mouse IgG (P0191, 1:200) and Alexa Fluor 647 goat anti-rabbit IgG (P0180, 1:200) (both from Beyotime Institute of Biotechnology). Next, cells were incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime Institute of Biotechnology) to label the cell nucleus. Negative control was performed by omitting the primary antibody. All incubation steps were followed by washes with PBS (10 min × 3 times). Cells were visualized and photographed using a confocal laser scanning microscope (Leica, Wetzlar, Germany). The mean fluorescence density was measured by Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).