Subcutaneous adipose tissue was removed and was fixed in 10% buffered neutral formalin. It was then embedded in paraffin, sectioned and stained. All adipose sections were viewed at 40X magnification, and images were captured using Zeiss microscope connected via camera to a computer (progres® capture pro 2.1 camera). Adipocytes size measurement was performed using a computer-assisted image analysis progres® capture pro software. From each animal 10 images were measured. The area of each adipocyte was measured by tracing the cell boundary on the images captured. All adipocytes that had complete cell boundary were measured. Data obtained after measurement were averaged to individual animal and later group mean and SEM were calculated.
For PRDM16 and UCP1 immuno staining, subcutaneous adipose sections were de-paraffinized and blocked in 1% BSA in PBS for 30 min at RT and incubated with PRDM16 (ProSci, USA) or UCP1(Abcam, USA) primary antibodies for 90 min at RT, followed by washing with PBS. Sections were then incubated with secondary antibody (goat anti-rabbit Alexa Fluor 555 secondary antibody, Molecular Probes, Invitrogen) for 30 min at RT and mounted using fluromount. Localization of PRDM16 and UCP1 were assessed and the images were captured at 40X magnification.
For PRDM16 and UCP1 immuno staining, subcutaneous adipose sections were de-paraffinized and blocked in 1% BSA in PBS for 30 min at RT and incubated with PRDM16 (ProSci, USA) or UCP1(Abcam, USA) primary antibodies for 90 min at RT, followed by washing with PBS. Sections were then incubated with secondary antibody (goat anti-rabbit Alexa Fluor 555 secondary antibody, Molecular Probes, Invitrogen) for 30 min at RT and mounted using fluromount. Localization of PRDM16 and UCP1 were assessed and the images were captured at 40X magnification.