Muscle Preparation

1. Male B6 X SJL F1 mice are allowed free access to food and water and kept on a 12/12-h light-dark cycle. The mice are then given an intraperitoneal injection of glucose (2 g/kg of body weight) and either insulin (6 U) or saline.

2. Over the next 30 min, the mice are anesthetized with pentobarbital (5 mg/100 g of body weight), and a butterfly needle connected to iv tubing is introduced in the dorsal tail vein of the animal. At exactly 30 min after the insulin or saline injection, 5% paraformaldehyde is injected into the tail vein and the animal is observed for whole-body fixation.

3. Immediately the hindlimb leg muscles are dissected for isolation of the soleus and dorsal tibialis muscles. Submerging the entire muscle in liquid nitrogen quickly freezes these fixed muscles.

4. An alternative method of muscle preparation is to anesthetize the mouse first and remove the right soleus or dorsal tibialis muscle for a basal state. This muscle is then fixed by incubation in 3% paraformaldehyde in PBS for 2 h. The mouse is then given glucose (2 g/kg of body weight) and insulin (6 U) by intraperitoneal injection. After 30 min, the remaining muscle of the pair is dissected and fixed as described in the preceding steps (see Note 6).

5. The frozen fixed or fixed muscle is then mounted on a cryostat holder with M-1 embedding matrix and cooled to -70°C. Frozen sections are cut with the cryostat at a thickness of 7-10 im and placed on Superfrost plus slides. The slides are stored at -70°C.

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