S3023

To detect adipocytes in the heart, thin myocardial sections were incubated in 4% formaldehyde solution, subjected to antigen retrieval in a sodium citrate buffer (pH 6.0), and incubated with an antibody against Perilipin 1 (PLIN1), a marker of lipid-containing adipocytes (Cell Signaling Technology, Cat# 9349), as published^{[24 (link)]}. Following overnight incubation, sections were treated with the secondary antibody conjugated to Alexa 594, and the nuclei were counterstained with 4’, 6 Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich St Louis, MO; Cat# D8417) at 1 μg/mL concentration. The stained sections were mounted using fluorescence mounting media (DAKO, Cat# S3023) and imaged using a Zeiss Axioplan fluorescence microscope. The total number of PLIN1-stained adipocytes were counted in each section, in 6 sections per mouse, and at least 5 mice per genotype. The average number of cells stained positive for PLIN1 expression per myocardial section was determined per group and compared among the genotypes.

Cells were fixed with 3% paraformaldehyde/5% sucrose for 15 min. Subsequently, cells were permeabilized with 50% methanol/50% acetone for 20 min at −20 °C and with 0.2% NP-40 for 10 min at room temperature. Cells were blocked with 5% normal goat serum (Wako)/5 mg/mL bovine serum albumin (BSA; Nacalai) for 15 min. As a mitochondrial marker, we used an OXCT1 antibody (Proteintech, 12175-1-AP), and a MTHFD2 antibody (abcam, ab56772) was used at 1:100 dilutions. Samples were reacted with the first antibody overnight at 4 °C. As secondary antibodies, goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (ThermoFisher Scientific, A-11004, 1:1000 dilution) and goat anti-rabbit IgG-FITC (Santa Cruz Biotechnology, sc-2012, 1:100 dilution) were used. Samples were reacted with secondary antibodies for 60 min. Finally, nuclei were stained with 0.2 μg/mL 4,6-diamidino-2-phenylindole (DAPI; Dojindo, 340-07971). Slides were mounted with fluorescence mounting medium (Agilent, S3023) and photographed by Zeiss LSM510 confocal microscopy.