Immunofluorescence Analysis of SERCA2a and Mfn2

SERCA2a and Mfn2 expression was assessed by immunofluorescence staining. Briefly, cells were collected and fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 10% goat serum albumin (Invitrogen, United States) for 1 h at room temperature (Vitturi et al., 2020 (link)). Subsequently, the samples were incubated with primary antibodies overnight at 4°C, washed with PBS three times, and incubated with secondary antibody for 45 min at room temperature. Finally, DAPI (Sigma-Aldrich, United States) was added, and the cells were analyzed under a fluorescence microscope.