Freeze Substitution Protocol for Muscle Samples

The samples were transferred to an EM AFS2 freeze substitution system (Leica Microsystems GmbH) and incubated using the following FS protocol either with ethanol absolute or acetone as solving agents: For 48 h, the muscle filets were incubated in ethanol absolute (Th. Geyer GmbH & Co. KG, Renningen, Germany) or anhydrous acetone (AppliChem GmbH, Darmstadt, Germany) at −90 °C. To efficiently remove residual water, the solution was refreshed after 24 h. After 48 h, the temperature was gradually raised to −60 °C within 15 h. At −60 °C, the samples were incubated with 2% PFA (AppliChem GmbH) in ethanol absolute [21 ] or anhydrous acetone, respectively, for 8 h. Within 18 h, the temperature was gradually raised to −30 °C, at which the samples were repeatedly washed with 2% PFA in either ethanol or acetone. After 17 h at −30 °C, the temperature was increased to 4 °C within 6 h. At 4 °C, the samples were washed with ethanol absolute/anhydrous acetone four times within 4 h, followed by a rehydration described as follows: In total, eight washings steps of 10 min each were performed with ethanol absolute/acetone in phosphate buffered saline (PBS) including 95%, 90%, 80%, 70%, 50%, 30% ethanol absolute/anhydrous acetone in PBS, and two rinses in 100% PBS.