Mouse anti hcn1

For protein isolation, human SAN and atrial tissue were homogenized in 2x urea buffer^{20 (link)} (10μl buffer per 1mg tissue) followed by centrifugation at 14,000 rpm for 10 min at 10°C, and the supernatant was collected. Protein yield was quantified using RCDC protein assay (Bio-rad). Equal amounts (20μg/sample) of proteins were separated by SDS-PAGE 12% (200:1) polyacrylamide gels and transferred to 0.45μM low fluorescence PVDF membrane by methods previously described^{20 (link)}. After blocking, membranes were incubated with various primary antibodies overnight at 4°C: mouse anti-HCN1 (1:500, Abcam), rat anti-HCN4 (1:100, Abcam), rabbit anti-HCN2 (1:500, Alamone), and rabbit anti-Cx43 (1:8,000, Sigma-Aldrich). Mouse anti-GAPDH (1:20,000, Sigma-Aldrich) was used as reference for equal protein loading and to normalize HCN channel protein band intensity. Subsequently, 1:2,000 diluted fluorescent DyLight conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were applied to membranes for 1 hour at room temperature. The specific bands were detected on a Typhoon 9410 imager (GE Healthcare) and quantified by densitometry analysis (ImageQuant, GE Healthcare).

Cryosections were fixed with −20°C methanol for 5min before immunostaining. Sections were permeabilized with 0.1% Triton X-100 (Sigma Aldrich), blocked with 1% bovine serum albumin, and incubated with primary antibodies overnight at 4°C. The following day, sections were incubated in secondary antibodies for 2 hours at room temperature and mounted in ProLong® Gold Antifade Mountant with DAPI (Life Technologies). The primary and secondary antibodies include: mouse anti-HCN1 (1:100, Abcam), rat anti-HCN4 (1:100, Abcam), rabbit anti-HCN2 (1:100, Alamone), rabbit anti-Cx43 (1:400, Sigma-Aldrich), mouse anti-α-actinin (1:200, Abcam), goat anti-rabbit Alexa Fluor 488 (1:200, Life Technologies), and goat anti-mouse Alexa Fluor 568 (1:200, Life Technologies). Paraffin sections were dewaxed and heated in citrate-based buffer for antigen retrieval before the immunostaining protocol. Sections were imaged using an Olympus FV1000 Filter confocal microscope and florescence density was measured by ImageJ software. A summary of antibodies used in immunohistochemistry protocols is provided in Supplemental Table I, and the specificity of antibody is discussed in Supplemental Material (Supplemental Figure I).