Histology and histomorphometry

Fixed murine and human soft tissue and decalcified bones (14% EDTA for 14 days) were paraffin embedded by the histology core at The University of Edinburgh’s Shared University Research Facilities (SuRF). Paraffin-embedded tissue sections were then sectioned at 100 µm intervals using a Leica RM2125 RTS microtome and collected onto 76 ×26 mm StarFrost slides (VWR, UK). The slides were baked at 37 °C overnight before Hematoxylin and Eosin (H&E) staining.

Quantification of multilocular adipocyte frequency was performed on H&E-stained iWAT and BMAT from male C57BL/6J mice aged ~18 weeks. Adipocytes were considered multilocular if they contained three or more smaller lipid droplets associated with a large lipid droplet, as previously described¹¹. Both the total marrow area and the number and area of adipocytes were measured and expressed as the number of Ads per marrow area (N.Ad/Ma.Ar; number/mm²) and adipocyte area as a percentage of marrow area (Ad.Ar/Ma.Ar; %) using image J⁶¹.