Mouse anti mitochondria

Immunohistochemistry was performed as described [40 (link), 41 (link)]. Briefly, tissue slides of BAT and inguinal fat were dewaxed in xylene, rehydrated in ethanol (100%, then 95%, ethanol washes) and rinsed in PBS. A heat-induced antigen retrieval step with Citric Acid Based Antigen Unmasking Solution (Vector laboratories, Burlingame, CA) was used to unmask antigens. To block endogenous peroxidases, slides were incubated in 3% H₂O₂ for 30 min at room temperature and then rinsed in PBS. Before primary antibody was applied, slides were soaked in blocking solution (containing 5% sheep serum, 0.2% BSA, and 0.1% Triton X-100 in PBS) for 1 h at room temperature. The following antibodies were used: rabbit-anti UCP-1 (1:50; Abcam) and mouse- anti mitochondria (1:25; Abcam). All antibody staining was performed at 4 °C overnight, followed by incubation with 1:1000 diluted anti-biotin secondary antibody (Vector Laboratories) for 45 min at room temperature. Slides were developed using a DAB kit (Vector Laboratories) and imaged using a DS-Fi1 camera connected to a Nikon E80i stereomicroscope. Images were processed using Nikon imaging software, NIS Elements RA3.2.