Biorevo microscope system

BAT was harvested, fixed overnight in 10% formalin, embedded in paraffin and sectioned for hematoxylin and eosin (HE) or immunofluorescence staining. The number of large lipid droplets (defined as droplets with a surface area >250 μm²) in the ×400 field was counted. For immunofluorescence, the sections were deparaffinized and retrieved with 10mM citrate buffer (pH 6.0). As the first antibody, anti-SIRT1 (Sigma-Aldrich, 07–131) was used, together with isolectin GS-IB4 (IB4, derived from Griffonia simplicifolia-biotin-XX conjugate) for staining of capillaries (Invitrogen, I21414), and Hoechst (Life Technologies, 33258). Secondary antibodies were Cy5 Goat anti-rabbit IgG (H + L; Life Technologies, A10523) for anti-SIRT1, BB515 streptavidin (BD horizon, 564453), and Cy5-streptavidin conjugate (Invitrogen, 43–4316) for IB4. TUNEL staining was performed to assess apoptosis. The sections were stained according to the instructions of the In situ Apoptosis Detection Kit (TaKaRa, MK500). HE-stained images were captured with a Biorevo microscope system (Keyence Co.), and immunofluorescence sections were assessed by confocal microscopy (NIKON, C2). ImageJ was used to quantify image analysis (Schneider et al., 2012 (link)).