Rabbit anti glut4 antibody

Tissues fixed in 4% formaldehyde were then embedded in OCT compound and cut into sections (thickness, 4 µm) according to a standard protocol. The sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope. For immunohistochemical staining of membrane-bound Glut-4, sections (thickness, 4 µm) were prepared from representative blocks of paraffin-embedded tissues, dewaxed, and rehydrated. Sections were then blocked with 3% H₂O₂ in PBS for 10 min at room temperature. To block non-specific binding, sections were incubated in 10% goat serum for 1 h. Sections were then incubated with rabbit anti-Glut4 antibody (Abcam, Cambridge, UK) at a dilution of 1∶4,000 in 10% goat serum overnight at 4°C. Goat serum replaced the primary antibody in negative controls. After several washes with PBS, sections were incubated with the Cy2-conjugated goat anti-rabbit secondary antibody (H&L, Alexa Fluor 488; Abcam) at a 1∶1,000 dilution for 1 h at room temperature. Sections were then counterstained with DAPI (4′6 diamidi-no-2-phenylindole, for nuclei staining), cover-slipped and examined under a fluorescence microscope.

Skeletal muscle from three rats in each group was randomly selected for GLUT4 immunofluorescence experiments. Briefly, after fixation in 4% paraformaldehyde overnight at 4 °C, the muscle tissues were equilibrated in 20% sucrose overnight at 4 °C until the tissues settled to the bottom. The tissues were then transferred into 30% sucrose overnight at 4 °C until they settled to the bottom, after which they were embedded in OCT for frozen sectioning. Sections were sliced at a thickness of 10 μm. Subsequently, frozen sections were rinsed gently with 4 °C phosphate-buffered saline (PBS) three times, fixed with 4 °C 4% PFA for 30 min at room temperature, and then washed three times with PBS again. After incubation with 0.2% Trition-X100 in PBS for 20 min, sections were blocked with 3% BSA for 30 min at room temperature and then incubated overnight with rabbit anti-GLUT4 antibody (1:250, Abcam, ab654, Cambridge, MA, USA) at 4 °C in a wet box. After washing with Tris-buffered saline containing 0.1% Tween 20, the sections were incubated with donkey secondary antibody conjugated with Alexa Fluor® 568 (1:400, Invitrogen, A-11011, Carlsbad, CA, USA) for 3 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA). All photomicrographs were taken using an inverted fluorescence microscope (Nikon Eclipse Ti-E, Nikon, Japan).