Oil red o

Lipid droplets in differentiating adipocytes were stained with Oil Red O (Sigma Aldrich, O0625).
Briefly, the differentiation medium was removed, and the cells were washed once with PBS (Gibco™, 14,190–0949) before fixing in 4% paraformaldehyde for 30 min. The cells were then incubated in 60% isopropanol for 5 min. The cells were subsequently washed with distilled water and incubated for 5 min with an Oil Red O staining solution (1:3 dilution of 0.3% Oil Red O in isopropanol and distilled water), followed by three washes with distilled water. All steps performed at room temperature. Images of Oil Red O-stained cells were taken with an inverted microscope (Carl Zeiss).
Oil Red O was extracted from the cells by removing the distilled water and adding 99% isopropanol. Following thorough mixing, the isopropanol containing the extracted Oil Red O was transferred to a 96-well plate. The absorbance was measured at 492 nm, subtracting the absorbance from the pure isopropanol used for extraction.

10-μm RV cryosections were stained with 0.5% Oil Red O (Sigma-Aldrich) in propylene glycol overnight and then counterstained with hematoxylin (Abcam) to identify intramyocardial lipid deposits. Sections were imaged with a Zeiss AxioCam IC and percent Oil Red O stain was determined using FIJI.

Briefly, the differentiated porcine pre-adipocytes were gently washed three times with fresh 1×PBS and then fixed in 4% paraformaldehyde for 30 min. The fixed cells were washed three times with 1×PBS and stained with 60% saturated oil red O for 30 min (Sigma-Aldrich, Shanghai, China). Subsequently, the fixed cells were washed three times with 1×PBS. Images of the cells were captured using a Zeiss Axiovert 40 CFL inverted microscope (Thornwood, NY, United States).
Total triglyceride was quantified by the elution of oil red O with isopropanol and the absorbance was measured at the 510 nm wavelength.