Ab50599

AC16 cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with 0.4% Triton-X at room temperature for 20 min. The samples were coincubated with Na_V1.5 (ASC-005, Alomone Labs, Israel, 1:200), Kir2.1 (APC-026, Alomone Labs, Israel, 1: 200), and α-actinin (ab50599, Abcam, Cambridge, United Kingdom, 1:100) antibody at 4°C overnight. Then the AC16 cells were incubated with the appropriate secondary antibody at room temperature for 2 h and the nuclei were stained with DAPI (Beyotime, Shanghai, China, 1:1,000) for 10 min. Immunofluorescence was observed under a confocal laser scanning microscope (Nikon 80i, Otawara, Tochigi, Japan).

Paraffin-embedded sections were 5-μm thick sections, deparaffinized with xylene and ethanol, and then heated in 0.1 mol/L citrate buffer (pH 6.0) by microwaving for 15 min. After being cooled at room temperature, the sections were incubated in 3% hydrogen peroxide for 20 min to block endogenous peroxidase. Then, the sections were blocked with 10% (v/v) BSA (Sangon, Shanghai, China) to inhibit non-specific binding, followed by incubation with primary antibody for ACTN1 (Abcam, ab50599) at 4 °C overnight with a dilution at 1:200. After washing with PBS for three times, sections were incubated with secondary reagent. All the slides were labeled with DAB substrate liquid (Cell signaling Technology, USA) and counterstained by hematoxylin, and photographed with a microscope (Carl Zeiss, USA). Scoring was conducted according to the intensity of positive staining and the proportion of stained tumor cells (score 0: 0–5%, 1: 6–30%, 2: 31–70%, and 3: > 71%. Scoring was evaluated by two independent investigators who were blinded to the clinical information. Disagreements were resolved by consensus.