Pa5 23052

Human fiber bundles were teased into individual fibers and transferred to wells in a 24-well plate containing PBS using fine forceps. FDB fibers were similarly incubated in PBS. Fibers were washed 3 × 10 min in PBS and incubated in blocking buffer 1% bovine serum albumin (Merck), 5% goat serum (16210-064, Gibco), 0.1% Na Azide (247-852, Merck), 0.04% Saponin (27534-187, VWR) for 1 hr. The muscle fibers were then incubated in blocking buffer containing primary antibodies overnight at 4°. The next day, the fibers were washed 3 × 10 min in PBS containing 0.04% Saponin and incubated in blocking buffer with Alexa 488 anti-rabbit or Alexa 568 anti-rabbit or anti mouse (Invitrogen) for 2 hr. Finally, the fibers were washed 3 × 10 min in PBS and mounted on glass slides in Vectashield (H-1000, Vector Laboratories) or imaged directly from the glass bottom dish. The following antibodies were used, raised in rabbit: GLUT4 (PA5-23052, Invitrogen), detyrosinated α-tubulin (AB48389, Abcam), Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

gWATs were directly fixed in 4% paraformaldehyde solution for overnight. Formalin-fixed paraffin-embedded sections (3 μm-thick) were deparaffinized and hydrated, and underwent antigen retrieval using heat-induced epitope retrieval methods. For GLUT4 immunostaining, tissue sections were fixed and permeabilized with 0.02% Triton X-100 in PBS (PBST) for 15 min. Slides were blocked with 5% BSA in PBST for 30 min. Next, the slides were washed once with PBS and probed with anti-GLUT4 antibody (PA5-23052, Invitrogen) at a dilution of 1:500 overnight in 3% BSA in PBS at 4 °C. The slides were then washed three times for 5 min each in 0.05% Tween 20 in PBS, after which they were incubated with Alexa 488-conjugated goat anti-rabbit IgG secondary antibody (A11008, Invitrogen) diluted 1:200 in PBS containing 3% BSA for 30 min at room temperature. Finally, the slides were mounted with a mounting medium (VECTASHIELD, H-1000, USA). Slides were viewed and photographed using an LSM-7700 confocal imaging system (Carl Zeiss, Germany).