Bz 8100 microscope

BAT specimens were fixed with 4% paraformaldehyde, rinsed with water, transferred into 30% sucrose in PBS at 4 °C until specimens sunk, and embedded in Optimal Cutting Temperature (OCT) matrix compound. OTC-embedded specimens were cut into 4 µm-thick sections and stained for 60 min at 37 °C in Oil red O solution followed by counterstaining with hematoxylin and eosin. Oil red O staining was carried out by Biopathology Institute Co., Ltd. (Oita, Japan). Microscopic examinations were performed using a BZ-8100 microscope (Keyence, Osaka, Japan).

Histological examination of WAT and liver was carried out under standardized methods. The tissues were dissected, fixed in 10% buffered formalin overnight, washed with PBS, and embedded in paraffin. Sections were stained with hematoxylin and eosin for histological analysis, and the slides were examined under a Keyence BZ-8100 microscope (Keyence Japan, Osaka, Japan).

Cardiomyocyte differentiation was performed in 96-well plates. Cells were rinsed with 100 μL of phosphate-buffered saline (PBS) containing 0.5 mM CaCl₂ and 0.5 mM MgCl₂ (PBS+/+) and fixed for 20 min with 100 μL of 4% paraformaldehyde. The cells were rinsed with 100 μL of PBS+/+, permeabilized with 100 μL of PBS+/+ containing 0.2% Triton X-100 (020-81152, Kishida Chemical), blocked for 60 min with 10 mg/mL bovine serum albumin (BSA, A8806-1G, Sigma-Aldrich), and incubated overnight with primary antibodies (25 μL) in an immunostaining buffer at 4 °C with shaking. The cells were then incubated with secondary antibodies in an immunostaining buffer for 1 h at 23–28 °C with shaking. The primary and secondary antibodies were used after dilution in the immunostaining buffer. Nuclei were stained with 20 µg/mL 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI,045-30361, Wako). The cells were then imaged using a BZ-8100 microscope (Keyence) and analyzed with Image J software (NIH, Bethesda). The antibody information is listed in Table S3.