Rm2016 microtome

Mouse muscle tissues were frozen in isopentane that had been cooled in liquid nitrogen. Wheat germ agglutinin (WGA) staining was performed using fluorescein isothiocyanate (FITC)–conjugated WGA (Sigma-Aldrich, #L4859). Quantification of cross-sectional area of the myofibers was performed with National Institutes of Health ImageJ software. Adipose tissues were fixed in 4% formaldehyde overnight at 4°C immediately after euthanasia, embedded in paraffin, and cut to 7-μm sections on slides using a Leica RM2016 microtome. H&E and Masson’s trichrome staining were conducted according to the standard protocol. For UCP1 IHC, brown adipose tissue sections were rehydrated and then boiled in 10 mM citric acid buffer, pH 6.0, at 95°C for antigen retrieval.  IHC was performed using the UltraSensitive SP (Rabbit) IHC Kit (Maxim) according to the manufacturer’s instructions using rabbit UCP1 antibodies (1:500; ab10983, Abcam). DAB Staining Kit (DAB-0031, Maxim) was used according to the manufacturer’s instructions. Livers of mice were embedded in Tissue-Tek OCT cryostat molds (Leica) and frozen at −80°C. Ten-micrometer-thick sections of livers were generated in a cryostat. Tissue sections were stained in 0.5% Oil Red O and counterstained with H&E.

Livers of mice were embedded in Tissue-Tek OCT cryostat molds (Leica) and frozen at −80°C. 10-μm thick sections of livers were generated in a cryostat. Tissue sections were stained in 0.5% Oil-Red-O and H&E (Sigma-Aldrich) according to the standard protocol. Adipose tissues were fixed in 4% paraformaldehyde overnight at 4°C immediately after sacrifice, embedded in paraffin, and cut to 7-μm sections on slides using a Leica RM2016 microtome. The slides were stained with H&E. Quantification of cross-sectional area of the adipocyte was performed with NIH ImageJ software. At least 30 fields from random sections of each mouse sample were quantified. For UCP1 immunohistochemistry (IHC), adipose tissue sections were rehydrated and then boiled in 10 mM citric acid buffer, pH 6.0, at 95°C for antigen retrieval. Immunohistochemistry was performed using the UltraSensitiveTM SP (Rabbit) IHC Kit (Maxim) according to the manufacturer’s instructions using rabbit UCP1 antibodies (ab10983; 1:500; Abcam). DAB Staining Kit (DAB-0031, Maxim) was used according to the manufacturer’s instructions for development.

Hamster lung tissues were fixed in 4% paraformaldehyde for 10 days and, due to BSL-3 laboratory protocols, remained in the laboratory for that duration. After fixation, tissues were put into a dehydration container inside a fume hood. The tissues were then dehydrated using a gradient alcohol regimen in a DIPATH Donatello dehydration machine, progressing from 75% alcohol to paraffin III. These tissues, now soaked in paraffin, were embedded using a Wuhan Junjie Electronics JB-P5 machine: Melted paraffin was poured into molds, tissues from the dehydration container were added, labels were attached, and samples were cooled at −20°C. After solidification, paraffin blocks were trimmed and then sectioned to 4 μm in thickness using a Leica RM2016 microtome. These sections were floated on a 40°C water bath in a KD-P slide warmer to smooth out, then collected on glass slides, and oven-dried at 60°C. For deparaffinization, sections underwent treatment with eco-friendly solutions from Seville Biotechnology, followed by absolute ethanol washes and a final rinse in distilled water. After this process, sections were primed for H&E staining or immunohistochemistry.