Isolectin gs ib4

After the mice were euthanized, the tumors were excised and fixed in formalin, embedded in paraffin, cut into 5-μm sections, dewaxed and hydrated, and stained with hematoxylin and eosin using a standard technique. The IHC staining procedures were as follows: Tissue sections were incubated for 10 min at room temperature in 3% hydrogen peroxide to block endogenous peroxidase. Slides were blocked and incubated with the following primary antibodies: anti-EGFR (Nichirei Biosciences), pEGFR (Cell Signaling Technology), pERK (Thr²⁰²/Tyr²⁰⁴) (Cell Signaling Technology), pAKT (Cell Signaling Technology), endomucin (eBioscience), and CD34 (Abcam). The slides were subsequently incubated with polymer-HRP anti-rabbit (Dako) or anti-mouse (Dako) secondary antibodies. Tissue staining was visualized using 4′,6-diamidino-2-phenylindole (DAPI). The slides were then counterstained with hematoxylin, dehydrated, and mounted. The coimmunofluorescence staining procedure for isolectin GS-IB4 (594) (Invitrogen) was as follows: After the same preprocessing for IHC staining as described above, slides were incubated with primary antibody against isolectin GS-IB4 (594) and subsequently incubated with the appropriate fluorescence-conjugated secondary antibodies; DAPI counterstaining was used to identify the nuclei.

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)).