Muscle Immunoblotting

1. Again, the PAP pen is used to draw a circle around the sections in order to create a well. The cells are permeabilized by adding a droplet of 0.1% Triton X-100 in PBS for 15 min and then washed by mouth pipet three times in PBS-BSA droplets for 10 min each.

2. The slides are then blocked with PBS-BSA containing 20% donkey serum for 1 h. After washing in the same fashion, a drop containing 20 ig/mL of polyclonal rabbit anti-mouse GLUT-4 antibody is added to the cells. This antibody is affinity purified on a protein-A column. This solution is left in place for either 1 h at room temperature or overnight at 4°C in a humidified chamber.

3. Following the primary antibody incubation, the slides are washed as described previously and the secondary antibody, FITC-labeled goat anti-rabbit, is added as a droplet to the cells at a concentration of 1 :80.

4. Following three 10-min washes, the nuclear stain, TOPRO-3 in PBS at 4 iM, is added for 20 min. After careful washing, a drop of Vectashield is added to the slide and a cover slip placed and sealed with nail polish. The slides are then viewed by confocal microscopy.

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