The adipose tissue was quickly removed from the mice, washed with normal saline, dried and then fixed with 10% formalin. After rapid removal of the adipose tissue, it was placed in 4% paraformaldehyde. It was then dehydrated in an ascending series of ethanol, and equilibrated with xylene, followed by embedding in paraffin and sectioning into 5–10 µm slices. Then, the samples were dewaxed with xylene and a descending series of ethanol. Continued sections were stained with both Mayer’s hematoxylin and eosin (HE).
Dried paraffin sections were dewaxed and hydrated and then closed with 1% BSA for 30 min, the closure solution was blotted dry on blotting paper. Each section was incubated with UCP1 antibody (abcam1:100) overnight at 4°C in a wet box, and washed 3 times with PBS. After aspirating the residual liquid, the sections were incubated with fluorescent secondary antibody (FITC1:100) for 1 h at room temperature in a wet box protected from light, then rinse 3 times with PBS, the excess liquid was aspirated. A small amount of anti-fluorescence quencher containing DAPI dropwise was added, the sample was covered with a coverslip, and stored in a wet box protected from light for observation under a fluorescence microscope for photographs.
Dried paraffin sections were dewaxed and hydrated and then closed with 1% BSA for 30 min, the closure solution was blotted dry on blotting paper. Each section was incubated with UCP1 antibody (abcam1:100) overnight at 4°C in a wet box, and washed 3 times with PBS. After aspirating the residual liquid, the sections were incubated with fluorescent secondary antibody (FITC1:100) for 1 h at room temperature in a wet box protected from light, then rinse 3 times with PBS, the excess liquid was aspirated. A small amount of anti-fluorescence quencher containing DAPI dropwise was added, the sample was covered with a coverslip, and stored in a wet box protected from light for observation under a fluorescence microscope for photographs.
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