Immunohistochemistry

Immunohistochemistry was performed on paraffin embedded tissues. Sections were mounted on Superfrost Plus adhesion slides (Thermo), then deparaffinized and rehydrated as described above. Antigen retrieval was performed by boiling the slides in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6) in a water bath for 30 min. The slides were cooled down to room temperature for 30 min. Slides were rinsed with PBS (GIBCO® DPBS) and incubated with blocking buffer (3% BSA in PBS) for 1 h at room temperature. All incubations were performed in a humidity chamber. The slides were incubated with the primary antibody (anti-UCP1, 1:1000) diluted in 1% BSA in PBS overnight at 4°C. After primary antibody incubation, the slides were washed with PBS for 3x 10 min and then incubated with goat-anti-rabbit HRP secondary antibody at a 1:500 dilution for 1 h at room temperature. After secondary antibody incubation, sections were washed with PBS for 3x 10 min. Staining was performed using abcam DAB kit following the manufacturer’s instructions. After DAB-staining, the slides were rinsed with PBS to stop the chromogenic reaction and counterstained with hematoxilin for 2 min. Slides were incubated under running tap water for 10 min to achieve bluing of the hematoxilin. Afterward, the slides were dehydrated and mounted using Eukitt. Microscopy was performed on a Nikon A1 Microscope equipped with a DS-U3 camera (Nikon).