Mitotracker red

Cells grown on poly-L-lysine-pretreated coverslips were fixed with 4% p-formaldehyde followed by washing with phosphate-buffered saline (PBS) and then subjected to permeabilization with 0.25% Triton X100 (Sigma-Aldrich, MO, USA). Cells were washed with PBS three times, blocked with 1% Bovine Serum Albumin (BSA) in PBS-T for 1 h, and incubated with polyclonal anti-UCP1 antibody (1:200 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, followed by three washes with PBS. Cells were then incubated with fluoresceine isothiocyanate (FITC)-conjugated antigoat secondary antibody (1:400 dilutions). 4,6-diamino-2-phenyl indole (DAPI) (Thermo Fisher Scientific, Boston, MA, USA) was used to stain nuclei of cells. Florescence images were captured using a confocal laser scanning microscope LSM700 (Carl Zeiss, Oberkochen, Germany). Analysis of images (control and curcumin-treated) was performed by software Zen 2009 (Carl Zeiss, Germany). For staining of mitochondria, Mito Tracker Red (1 mM, Cell Signaling Technology, Beverly, MA) was directly added to the growing media at a concentration of 20–25 nM, and cells were kept for 30–40 min at 37°C. After incubation, cells were fixed in 4% p-formaldehyde, followed by a single wash with PBS and immunostaining (4 (link)).

HUVECs (5 × 10⁴cells/cm²) were cultured in a confocal dish for 24 h and then treated with various interventions. Subsequently, the treated HUVECs were stained with MitoTracker Red (Cell Signaling Technology, # 8778S, USA) for 30 min at 37 ℃ and fixed with cold methanol for 15 min at – 20 ℃. After undergoing 3 washes with PBS, they were permeabilized in Triton X-100 (Sigma-Aldrich, # T8787, USA) and blocked with 1%BSA, 10% goat-serum, and 0.3% glycine for 1 h at RT. Then, the cells were incubated with the primary antibody against LC3 (1:200) (Cell Signaling Technology, #4108S, USA) at 4 ℃ overnight. Subsequently, they were incubated with the related secondary antibody. Confocal images were captured with Leica SP5 confocal microscope (Leica, Germany) and analyzed by Fiji ImageJ software.

After euthanasia, sWAT and visceral WAT (vWAT) were rapidly collected and fixed in 4% paraformaldehyde solution for 48 h. The tissues were then paraffin-embedded and the resulting blocks were cut into 5–10 µm sections and stained with hematoxylin and eosin (H&E) to assess adipose histology. Photomicrographs were obtained using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan).
For immunofluorescence analysis, fixed cells and WAT sections were stained with rabbit anti-UCP1 (dilution, 1:200) or anti-CPT1 (dilution, 1:1000) antibodies overnight at 4 °C in a moist chamber. Fluorescein isothiocyanate (FITC)-conjugated (dilution, 1:1000) and Alexa Fluor™ 594-conjugated (dilution, 1:1000) secondary antibodies were used. Mitochondria were identified by staining with 1 mM MitoTracker Red (Cell Signaling Technology, Danvers, MA, USA), according to the manufacturer’s protocol, and nuclei were stained using DAPI (Thermo Fisher Scientific, Waltham, MA, USA) fluorescence. After mounting using ProLong Gold Antifade reagent (Thermo Fisher Scientific), fluorescence images were captured using a Zeiss confocal laser scanning microscope (LSM880; Carl Zeiss, Oberkochen, Germany) and Zen 2012 software (Carl Zeiss).

The changes in mitochondrial dynamics were detected by using MitoTracker Red CMXRos in B2B cells. After treatment with PQ or OAA for 48 h, cells were incubated with 100 nM MitoTracker Red (#9082, Cell Signaling Technology) for 30 min at 37°C. Then, cells were fixed with 4% paraformaldehyde for 10 min at −20°C and the nuclei were stained with DAPI (Beyotim Biotechnology), Finally, subsequent images were obtained by fluorescence microscope.