Superfrost plus slat

FD and TA muscles were cut into 50 μm-thick longitudinal slices using a cryostat (Leica Microsystems^®, Wetzlar, Germany), collected on glass slides (Superfrost Plus Slat^®, Thermo Scientific^®, Waltham, MA, USA), rehydrated in a phosphate buffer solution (PBS), and then immersed in PBS containing 10% normal donkey serum (NDS) and 0.2% Triton X-100. Sections were then incubated overnight in the same blocking solution with a rabbit anti-activated caspase 3 (1/1000, Cell Signaling Technology^®, Beverly, MA, USA). Following several washes in PBS, sections were incubated for 2h in PBS containing 5% NDS with Alexa Fluor^® 488 donkey anti-rabbit antibody (1/400, Invitrogen^®, Carlsbad, CA, USA). Activated caspase 3 positive cells were quantified in muscles using an epifluorescence microscope (Leica Microsystems^®) to determine the apoptosis rate as the average number of dead cells per area of 0.3 cm².

FD and TA muscles were cut, on their entire length, into 50 μm-thick longitudinal slices using a cryostat (Leica Microsystems^®, Wetzlar, Germany), collected on glass slides (Superfrost Plus Slat Thermo Scientific, Waltham, USA), rehydrated in a phosphate buffer solution (PBS), then blocked with PBS containing 10% normal donkey serum (NDS) and 0.2% Triton X-100. Approximately, 400 sections per muscle were then incubated overnight in the same blocking solution with a rabbit anti-activated caspase 3 (1/1000, Cell Signaling Technology^®, Beverly, USA). Following several washes in PBS, sections were incubated for 2h in PBS containing 5% NDS with Alexa Fluor^® 488 donkey anti-rabbit antibody (1/400, Invitrogen^®, Carlsbad, USA). Activated caspase 3 positive cells were quantified in muscles using an epifluorescence microscope (Leica Microsystems^®, Wetzlar, Germany) to determine the apoptosis rate as the average number of dead cells per area of 0.3 cm².