Mice were anesthetized with 2% isoflurane via nose cone while lying on a heated bed. For skeletal muscle fixation, hindlimb skin was peeled back and hindlimbs were immersed in fixative solution (2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2) for 30 minutes in vivo. For cardiac fixation, the chest cavity was opened, and the heart was perfusion fixed with a syringe attached to a 30 G needle through the apex of the left ventricle slowly pushing 2 ml of relaxing buffer (80 mM potassium acetate, 10 mM potassium phosphate, 5 mM EGTA, pH 7.2) followed by 2 ml of fixative solution. After initial fixation, the tissues were excised, cut into 1 mm3 (link) cubes, and placed into standard fixative solution (2.5% Glutaraldehyde, 1% Paraformaldehyde, 0.12 M sodium cacodylate, pH 7.2–7.4) for 1 h.
Samples were post-fixed and stained en bloc using an established protocol with minor modifications46 (link),47 (link). After five washes (always 3 min each) with 0.1 M cacodylate buffer at room temperature, samples were post-fixed in reduced 4% osmium solution (3% potassium ferrocyanide, 0.2 M cacodylate, 4% aqueous osmium) for 1 h on ice, washed five times in bi-distilled H2O, and incubated in fresh thiocarbohydrazide solution for 20 min at room temperature. In a second post-fixation step, samples were incubated in 2% osmium solution for 30 min on ice and washed five times in bi-distilled H2O. The sample was then incubated in 1% uranyl acetate solution and left in a refrigerator (4 °C) overnight, washed five times in bi-distilled H2O, incubated at 60 °C for 20 min with Walton’s lead aspartate (0.02 M lead nitrate, 0.03 M aspartic acid, pH 5.5), and washed five times in bi-distilled H2O at room temperature. The sample was next dehydrated in a graded ethanol series (20%, 50%, 70%, 90%, 95%, 100%, and 100%; 5 min each), and they were incubated in 50% Epon (50% ethanol) for 4 h and incubated in 75% Epon resin (25% ethanol) at room temperature overnight. Next day samples were incubated in fresh 100% Epon resin in one, one, and 4 h in order. After removing excess resin using filter paper, the Samples were placed on aluminum Zeiss SEM Mounts (Electron Microscopy Sciences, #75510) and polymerized in a 60 °C oven for 2 days. After polymerization, stubs were mounted in a Leica UCT Ultramicrotome (Leica Microsystems Inc., USA) and faced with a Trimtool 45 diamond knife (DiATOME, Switzerland) with a feed of 100 nm at a rate of 80 mm/s.
Samples were post-fixed and stained en bloc using an established protocol with minor modifications46 (link),47 (link). After five washes (always 3 min each) with 0.1 M cacodylate buffer at room temperature, samples were post-fixed in reduced 4% osmium solution (3% potassium ferrocyanide, 0.2 M cacodylate, 4% aqueous osmium) for 1 h on ice, washed five times in bi-distilled H2O, and incubated in fresh thiocarbohydrazide solution for 20 min at room temperature. In a second post-fixation step, samples were incubated in 2% osmium solution for 30 min on ice and washed five times in bi-distilled H2O. The sample was then incubated in 1% uranyl acetate solution and left in a refrigerator (4 °C) overnight, washed five times in bi-distilled H2O, incubated at 60 °C for 20 min with Walton’s lead aspartate (0.02 M lead nitrate, 0.03 M aspartic acid, pH 5.5), and washed five times in bi-distilled H2O at room temperature. The sample was next dehydrated in a graded ethanol series (20%, 50%, 70%, 90%, 95%, 100%, and 100%; 5 min each), and they were incubated in 50% Epon (50% ethanol) for 4 h and incubated in 75% Epon resin (25% ethanol) at room temperature overnight. Next day samples were incubated in fresh 100% Epon resin in one, one, and 4 h in order. After removing excess resin using filter paper, the Samples were placed on aluminum Zeiss SEM Mounts (Electron Microscopy Sciences, #75510) and polymerized in a 60 °C oven for 2 days. After polymerization, stubs were mounted in a Leica UCT Ultramicrotome (Leica Microsystems Inc., USA) and faced with a Trimtool 45 diamond knife (DiATOME, Switzerland) with a feed of 100 nm at a rate of 80 mm/s.
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