Thermo 6502

Fresh tissues were fixed in 4% paraformaldehyde for 24 hours at RT and then embedded in paraffin. Sections (5 μm thick) were used for UCP1 (1:200) (#14670, Cell Signaling) immunocytochemistry (ICC) staining or hematoxylin and eosin (H&E) staining in iBAT and ingWAT as previously described (16 (link)). In addition, 5-μm-thick sections from placenta at E18.5 were used for apelin (1:50) (#11497-1-AP, Proteintech) ICC staining. Goat anti-mouse IgG2b Alexa Fluor 488 secondary antibody (#A-21141; 1:250; Thermo Fisher Scientific, Waltham, MA) and anti-rabbit IgG Alexa Fluor 488 secondary antibody (#4412; 1:250; Cell Signaling Technology) were used. The cross-sectional areas of lipid droplet were measured using ImageJ software (National Institutes of Health, Bethesda, MD). For Oil Red O staining, liver samples from the offspring were fixed in PBS containing 4% paraformaldehyde and embedded in optimal cutting temperature compound (Thermo 6502, Thermo Fisher Scientific). Sections at 10-μm thickness were stained, and images were obtained by EVOS XL Core Imaging System (Mill Creek, WA).

For immunocytochemical (ICC) staining, skeletal muscles were removed and embedded in paraffin following 24 h fixation with 4% paraformaldehyde. Sections (5 μm thickness) of soleus and tibialis anterior muscles were prepared for CD31 (dilution 1:50) ICC staining as previously described (Son et al., 2017 (link)). For the secondary antibody, anti-rabbit IgG Alexa 555 (dilution 1:200) was used.
For staining specific muscle fiber types, fresh tissues were embedded in OCT compound (Thermo 6502, Thermo Fisher Scientific, Waltham, MA, USA), and sections (10 μm thickness) were utilized for staining using anti-myosin heavy chain (MHC) I, anti-MHC IIa, and anti-MHC IIb mouse monoclonal antibodies as primary antibodies. For the secondary antibodies, goat anti-mouse IgG2b Alexa 488, goat anti-mouse IgG1 Alexa 555, and goat anti-mouse IgM Alexa 350 antibodies were purchased from ThermoFisher Scientific. Images were generated by the EVOS® XL Core Imaging System (Mil Creek, WA, USA). Then, the cross-sectional areas were measured in ImageJ (NIH) according to the previous report (Son et al., 2020 ).