*2.7. Hystological Analysis*

Samples of visceral WAT (mesenteral), liver, and gastrocnemius skeletal muscle were fixed by immersion in 4% formaldehyde in 0.1 M phosphate buffer (overnight at 4 ◦C). The samples were dehydrated in ethanol, cleared, and then embedded in paraffin blocks. The tissues were cut into serial 6-πm-thick sections and then stained with hematoxylin-eosin for morphological examination. For adipocyte size quantification, evaluations were performed on three different hematoxylin-eosin slides (sections every 400 μm) for each animal and at least 400 adipocytes per animal were analyzed. The sections were viewed with a Nikon Eclipse 80i light microscope (Nikon Instruments, Milan, Italy) at 20× magnification. Images were obtained with a Sony DS-5M camera connected to an ACT-2U image analyzer. The mean surface area and the frequency distribution were calculated from at least four mice for each group, adipocyte size distribution is presented as the percentage of the total amount of cells.

#### *2.8. Body Composition and Energy Gains*

Body composition and carcass energy content were evaluated, as reported by Iossa et al. [29]. In brief, after removing the gu<sup>t</sup> contents, the carcasses were autoclaved and homogenized in water. The water content of the carcass was detected by drying the homogenate at 70 ◦C in a vacuum oven. Small pellets of dried homogenate were then used to evaluate the carcass energy content by bomb

calorimetry. Other homogenate aliquots were used to detect the lipid content by the Folch et al. method [30]. Body protein content was determined from a general formula relating to the total energy value of the carcass, the energy derived from fat, and the energy derived from protein [31,32]. The caloric values for body fat and protein were taken as 38.6 and 22.7 kJ/g, respectively. To detect lipid and protein gains, as well as energy e fficiency in WT and UCP3 KO mice, six mice from each genotype were euthanatized at weaning (i.e., when they were 24 days old) that corresponded to the beginning of dietary treatments (groups were named WT-time 0 and KO-time 0). Two additional groups of mice for each genotype were individually caged for 80 days since weaning, and feed ad libitum with either a STD diet or a HFD, as described above (groups were named WT-STD, KO-STD, WT-HFD, KO-HFD). The duration of the treatment with the high fat diet was chosen, since it was long enough to induce a HFD induced obesity in mice that were acclimated at thermoneutrality [33]. During the treatment, the body weight and food intake of the mice were monitored twice weekly. Feed spillage was taken into account when calculating the energy intake during the treatment. Body composition and energy content were evaluated, as described above. Carcass total energy gain, as well as the amount of energy that is gained and stored as lipids or as proteins after 80 days of either STD or HFD were determined by subtracting the total carcass energy, the carcass lipid-derived energy, and the carcass protein-derived energy detected in the WT-time 0 and KO-time 0 groups from the respective values detected in the WT-STD, WT-STD, KO-STD, and KO-HFD groups. The e fficiencies of energy, lipid, and protein deposition were calculated as: (energy gain/energy consumed by diet) × 100, (lipid gain/lipid-derived energy consumed by diet) × 100, (protein gain/protein-derived energy consumed by diet) × 100, respectively.
