Immunofluorescence Assay of HCN4 Expression

Immunofluorescence assay was performed as described previously (8 (link), 21 (link)). At 36, 48, and 72 h after transfection, HEK293T cells were grown on microscope slides and fixed in 4% preheated paraformaldehyde for 10 min. The fixed cells were rinsed three times with PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 15 min, blocked for 1 h in 10% goat serum, and then incubated with HCN4 antibody (Abcam) overnight at 4 °C. The cells were then incubated with an Alexa Fluor 488-conjugated goat anti-rat antibody (Invitrogen) for 1 h in the dark and washed with PBS before DAPI staining and mounting in the dark. The cells were analyzed by fluorescence confocal microscopy (Leica). Identical parameters were used for image acquisition and analysis. A threshold was set for each image to eliminate background.