All the tissues were fixed in 4% neutral formaldehyde for 24 h at room temperature, followed by dehydration, paraffin embedding, sectioning (4 mm), and staining with hematoxylin and eosin (H&E). To assess the fatty infiltration of skeletal muscle, tissues were snap frozen in liquid-nitrogen-cooled isopentane, sectioned at a thickness of 10 μm with a cryostat, and then stained with Oil Red O as described previously (19 (link)).
The procedure for immunohistochemistry was as previously described (20 (link)). Briefly, non-specific binding sites were blocked using 1% bovine serum albumin, followed by epitope retrieval using an autoclave (15 min in citrate buffer, pH 6.0). Then, the slides were incubated overnight at 4°C with rabbit polyclonal anti-UCP1 primary antibody (ab234430; Abcam) diluted 1:1000. After the slides were rinsed, they were incubated with HRP-conjugated goat anti-rabbit IgG (HAF008; R&D). Immunovisualization was carried out by the use of diamino-benzidine cytochemistry. The slides were finally counterstained with hematoxylin to display the nuclei. Indirect calorimetry and aerobic testing. Energy expenditure was analyzed using a Comprehensive Lab Animal Monitoring System (Columbus Instruments, United States). Pictures were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, United States). All pictures were assessed by two blinded reviewers.
The procedure for immunohistochemistry was as previously described (20 (link)). Briefly, non-specific binding sites were blocked using 1% bovine serum albumin, followed by epitope retrieval using an autoclave (15 min in citrate buffer, pH 6.0). Then, the slides were incubated overnight at 4°C with rabbit polyclonal anti-UCP1 primary antibody (ab234430; Abcam) diluted 1:1000. After the slides were rinsed, they were incubated with HRP-conjugated goat anti-rabbit IgG (HAF008; R&D). Immunovisualization was carried out by the use of diamino-benzidine cytochemistry. The slides were finally counterstained with hematoxylin to display the nuclei. Indirect calorimetry and aerobic testing. Energy expenditure was analyzed using a Comprehensive Lab Animal Monitoring System (Columbus Instruments, United States). Pictures were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, United States). All pictures were assessed by two blinded reviewers.
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