Permeabilization buffer

Adipose and liver tissues were fixed in 10% formalin for 12–24 h, embedded in paraffin, and sectioned. Sections were stained with Hematoxylin and eosin (Beyotime, C0105S) according to the manufacturer's instructions. The images were acquired by optical microscope (Nikon) using a 20× objective. Adipocyte sizes were quantified by ImageJ and AdipoCount. For UCP1 IHC staining, 5µm‐ thick iWAT sections were pre‐incubated with permeabilization buffer (Beyotime) for 30 min at room temperature and then incubated with UCP1 antibody (Abcam, ab10983) in blocking buffer (5% normal goat serum in PBS) at 4 °C overnight. Antigens were detected using primary antibody and in conjunction with a DAB chromogenic detection kit (ZSGB‐BIO, ZLI‐9018) according to the manufacturer's instructions. Immunostained images were collected on optical microscope (Nikon).

For the proliferation of hASMCs, we utilized Edu-594 kit C0078S, Beyotime, China). hASMCs (1 × 10⁴/well) were appended to twelve-pore plates containing coverslips. After adhesion, cells were intervened based on the above study design for 48 h. Thereafter, 2 × EdU solution was injected into each well (37°C, 4 h). Then, 95% ethanol was employed to fix cells (37°C, 15 min). After rinsing, permeabilization buffer (P0097, Beyotime, China) was added (37°C, 10 min). After rinsing again, the Click solution was added (37°C, 0.5 h, darkroom). DAPI was exploited to identify the nucleus (37°C, 10 min, dark room). For the observation of the proliferative ability, a fluorescence microscope was used.

On the preceding day, human- and murine-derived colorectal cancer cell lines including MC38, RKO, HCT15, HCT116, SW620 and DLD1 were planted in 4-well confocal dishes, with each well receiving a density of 1 × 10⁵ cells. The subsequent day, post-seeding, the cells underwent a rinse with phosphate-buffered saline (PBS), followed by fixation in 4% paraformaldehyde at room temperature for 20 min. After fixation, permeabilization was carried out using permeabilization buffer (Beyotime Biotech, Shanghai, China) for 20 min, succeeded by a 30-min blockage with immunostaining blocking buffer (Beyotime Biotech, Shanghai, China). Following this, the samples were subjected to an overnight incubation at 4 °C with the primary antibody pan-kla (#PTM-1401RM, PTMBio, Hangzhou, China) at a 1:100 dilution in blocking buffer. Subsequent to thorough washing, the cells were treated with fluorescent-labeled secondary antibodies for 1 h at room temperature. DAPI staining (Sigma, USA) was applied, and the observation was conducted using a confocal microscope (Olympus, Japan).