Na azide

GLUT4 translocation was measured as recently described^{43 (link)}. Briefly, rapidly dissected TA muscles were fixed by immersion in ice-cold 4% in paraformaldehyde in PBS for 4 h. TA muscles were transferred to a Sylgaard dish and isolated into smaller bundles using fine forceps. The bundles were transferred to a storage solution containing PBS and glycerol (1:1) and kept at 4 °C overnight. Individual fibers were teased from fixed muscle with fine forceps under a dissection microscope. Non-permeabilized isolated muscle fibers were incubated in blocking buffer (1% BSA, 5% goat serum (16210-064, Gibco), 0.1% Na Azide (247-852, Merck) for 1 h and then incubated with an anti-myc antibody overnight (Supplementary Table I). The next day, fibers were then washed 3 × 10 min in PBS containing 0.04% saponin and incubated with Alexa 568 antibody (Supplementary Table I) in blocking buffer containing 0.04% saponin for 120 min Individual muscle fibers were transferred to mounting media (H-1000; Vector Laboratories) on a glass slide and covered by a coverslip. A minimum of 10 transfected fibers were blindly imaged from each muscle (a total of 60 fibers) using a ×63 1.4 NA Plan-Apochromat objective on a Zeiss 780 microscope driven by Zeiss Zen Black 2012. Image analysis is described in the image analysis sections and as previously described^{43 (link)}.

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).

Primary and secondary antibodies are listed in S1 Table. At desired stage of development, mouse eyes were collected and fixed 5 h in ice-cold 2% paraformaldehyde (PFA) in PBS for 5 h at 4°C. Thereafter retinas were dissected in PBS. Blocking/permeabilisation was performed using Claudio’s Blocking Buffer (CBB), consisting of 1% FBS (Gibco), 3% BSA (Sigma), 0.5% triton X100 (Sigma), 0.01% Na deoxycholate (Sigma), 0,02% Na Azide (Sigma) in PBS pH = 7.4 for 2–4 h at 4°C on a rocking platform. Primary and secondary antibodies were incubated at the desired concentration in 1:1 CBB:PBS at 4°C overnight in a rocking platform. When using two rabbit primary antibodies, such for Erg and Golph4 immunofluorescence images in Figs 3, 5, S4, and S5, after first incubation with Erg primary and corresponding secondary, an additional step of incubation with donkey anti-rabbit Fab fragments (1:100, Jackson’s Laboratories) followed by 15 min fixation with 4% PFA was performed prior the incubation with Golph4 primary antibodies, in order to avoid intensive cross-reaction between the two primary antibodies. DAPI (Sigma) was used for nuclei labeling. Retinas were mounted on slides using Vectashield mounting medium (Vector Labs, H-1000). For imaging we used a Carl Zeiss LSM780 scanning confocal microscope.