Adipose tissue was collected, embedded in paraffin, and stained as previously described (Holtrup, et al. 2017 (link); Jeffery et al. 2015 (link); Jeffery et al. 2016 (link); Sebo and Rodeheffer 2021 ). For adipocyte nuclei analysis, 20–30 images for every tissue section were acquired at 40X with a Leica TCS SP5 confocal microscope. Quantification of BrdU in adipocyte nuclei was done as previously described (Jeffery et al. 2015 (link)). At least 50 adipocyte nuclei were scored for each animal.
For adipocyte diameter measurements, the area of each adipocyte (in square pixels) was measured using Cell Profiler. The diameter of each adipocyte was calculated using the measured area, assuming each adipocyte is a perfect circle. At least 200 adipocytes were measured for each animal.
For whole mount microscopy, tissues were dissected and cut into ~1.5×1.5 cm pieces. Samples were subsequently mounted onto microscope slides with Fluoromount-G (SouthernBiotech, 0100–01) and imaged at 20X with a Leica TCS SP5 confocal microscope.
For adipocyte diameter measurements, the area of each adipocyte (in square pixels) was measured using Cell Profiler. The diameter of each adipocyte was calculated using the measured area, assuming each adipocyte is a perfect circle. At least 200 adipocytes were measured for each animal.
For whole mount microscopy, tissues were dissected and cut into ~1.5×1.5 cm pieces. Samples were subsequently mounted onto microscope slides with Fluoromount-G (SouthernBiotech, 0100–01) and imaged at 20X with a Leica TCS SP5 confocal microscope.
