Ab66501

Whole-mount IHC was performed as described previously (30 (link), 68 (link)). The following primary antibodies were used: (a) anti-HCN4 (Abcam, ab66501, 1:200 dilution), a polyclonal antibody raised against rat HCN4, and (b) anti-AC_I (Santa Cruz Biotechnology Inc., sc-365350, 1:100 dilution), a monoclonal antibody raised against mouse AC_I. SAN tissue was washed with PBS (3 × 10 minutes) and then incubated with anti-rat and anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, 1:1,000 dilution) for 4 hours at room temperature in the dark. It was then washed in PBS (3 × 10 minutes) and incubated for 2 hours with 20% DMSO diluted in PBS. Coverslips were mounted on the slides with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). The slides were sequentially imaged using a Zeiss 900 confocal laser-scanning microscope equipped with an Airyscan detector module, a Plan-Apo 63× 1.4 NA oil-immersion objective, and 488/561 lasers. Imaris software (Bitplane) was used to perform 3D reconstructions.

As previously described [11 (link), 12 (link)], heart tissue samples were embedded in paraffin and sectioned (4 μm) before immunohistochemical analysis. After dewaxing and debenzolization, rabbit anti-HCN4 antibody (ab66501; Abcam, USA) was added to the sections at a dilution of 1:200 at 4°C overnight, followed by washing with tris buffered saline (TBS) at room temperature, with subsequent addition of anti-rabbit horseradish peroxidase (Beijing Zhongshan Golden Bridge Biotechnology Co.) at 37°C for 2 h. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted using neutral gum.