10 µm serial cuts were performed on the same tissue samples embedded in paraffin used for H&E staining. UCP1, PGC1 alpha and HSL expression were studied by means of immunohistochemistry. Briefly, hRAN and hRAT microtome slides were first deparaffinized, and then a heat-mediated antigen retrieval, endogenous peroxidase blocking and nonspecific tissue blocking were performed. Slides were then incubated with the different primary antibodies (Anti-UCP-1. SIGMA U6382. Dilution of 1:500; Anti-PGC1 alpha. Abcam ab54481. Dilution of 1:300; and Anti-HSL. Abcam ab45422. Dilution of 1:300) at 4 °C. And after that with an anti-rabbit biotinylated secondary IgG antibody. Finally, slides were incubated with peroxidase-conjugated streptavidin. Peroxidase reaction was performed with chromogen 3,3′-diaminobenzidine (DAB) (DAKO LSAB + Kit, HRP). Hematoxylin counterstaining was performed. Serial cuts incubated in the absence of the primary antibody were used as negative controls. Images were taken with a Nikon Eclipse E200 Microscope fitted with a Micrometric SE Premium (Nikon Corp., Japan) digital still camera at 10× and 40× magnification. DAB staining quantification in the three tissue types was performed in 5 fields of each preparation as mentioned above3 (link)–5 (link).
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