Immunohistochemical Analysis of UCP1 in Adipose Tissue

Samples were collected and fixed with 4% paraformaldehyde in PBS. After fixation, tissues were embedded in paraffin, before cutting into 4 µm sections and staining with hematoxylin and eosin (H&E) or performing immunohistochemistry (IHC) with rabbit anti-UCP1 antibody (Abcam, ab10983)^{6 (link)}. For IHC, rehydrated sections were microwaved in 10 mM citric acid buffer (pH 6.0) for antigen retrieval, and endogenous peroxidases were quenched with BLOXALL Blocking solution (Vector Laboratories, Burlingame, California). Sections were blocked with Avidin D, biotin and protein blocking agent in sequential order followed by application of the anti-UCP1 antibody (1:500). A biotinylated anti-rabbit antibody was used as a secondary antibody. Horseradish peroxidase-conjugated ABC reagent was applied, and then DAB reagent was used to develop the signal before counterstaining in hematoxylin and dehydrating the sections in preparation for mounting. Stained sections were acquired using a Leica DME microscope and Leica ICC50HD camera (Leica, Wetzlar, Germany) and analyzed using Leica LAS EZ software. Adipocyte size quantitation was performed using the Adiposoft plugin of ImageJ (NIH, Maryland, USA).