Ab69054

Immunofluorescence was performed according to previously reported methods (10 (link)). Transfected cMSCs (1.5×104 cells/cm2) were fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 15 min at room temperature, washed in phosphate-buffered saline (PBS; AR0030; Wuhan Boster Biological Technology, Ltd.), then treated with 0.2% Triton X-100 (T8787; Sigma-Aldrich) for 15 min. Cells were incubated with rabbit polyclonal anti-HCN4 antibody (1:100; ab69054; Abcam, Cambridge, UK) overnight at 4°C. Following washing three times with PBS for 5 min, the cells were incubated with Alexa FluorTM 647-conjugated donkey anti-rabbit IgG (1:200; A-31573; Invitrogen) for 60 min at 25±1°C. After further washing with PBS, the cells were mounted with Antifade Mounting Medium (Beyotime Institute of Biotechnology, Shanghai, China). Nuclei were stained with 4′,6-diamidino-2-phenylindole (D9542; Sigma-Aldrich) as a location control. Fluorescent images were obtained using an inverted laser confocal microscope (LSM 710; Carl Zeiss Microscopy GmbH, Cologne, Germany). The results were analyzed using ZEN lite software, 2011 edition (Carl Zeiss Microscopy GmbH).

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).