Mouse anti α actinin 2

Indirect immunofluorescence was performed on 10 μm control (GFP) Na_v1.5-WT or R104W-injected mouse ventricle cryosections fixed with paraformaldehyde for 15 min. Sections were washed twice for 5 min with phosphate buffer saline (PBS), blocked in PBS-5% bovine serum albumin (BSA) for 30 min at room temperature. Sections were then incubated overnight with primary antibodies at 4°C: the rabbit anti-GFP (1:2000, Torrey Pines Biolabs, United States) to detect exogenous hNa_v1.5-GFP, and mouse anti-α-actinin 2 (1:500, Sigma-Aldrich, United States). Heart sections were then washed twice with PBS and incubated 1 h with secondary antibodies: goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 (1:1000, Molecular Probes, Thermo Fisher Scientific, United States), and the nuclear dye DAPI (1:2000, Merck, Germany) diluted in the blocking buffer. Control experiments were performed by omitting primary antibodies.
Labeled ventricle sections were observed with a DeltaVision epifluorescent microscope (20× or 60×). Images were analyzed with DeltaVision imaging system (GE Healthcare, Seattle, WA, United States) equipped with 3D-deconvolution. For each sample, series of consecutive plans were acquired (sectioning step: 0.2 μm).