Leica cm1850uv

Female mice were anesthetized with ketamine-xylazine and perfused intracardially with saline (0.9% NaCl) followed by 4% PFA in PBS (pH 7.4). Fixed brains were immersed in 30% sucrose and 0.01% sodium azide in PBS at 4 °C for 2 days. Next, 3 sets of coronal sections (30-μm-thick) were cut in a freezing microtome Leica CM1850 UV (Wetzlar, Germany) and stored at −20 °C in cryo-protectant. One set of free-floating sections from each animal was washed in TBS 0.1 M and incubated in blocking solution (2% donkey serum + 0.3% Triton X-100) in TBS 0.1 M for 60 min. Then, sections were incubated with rabbit antibody against green fluorescent protein (GFP) (Abcam ab290) in blocking solution overnight at 4 °C. For GFP visualization, we used Cy3 donkey anti-rabbit (Jackson ImmunoResearch Labs, 711-165-162). Sections were then washed and coverslipped with Fluorogel mounting solution. Images were captured with fluorescence stereo microscopes Leica M205 and camera CCD Leica 7000 T.

Wild-type (C57BL/10J, The Jackson Laboratory, Bar Harbor, ME, USA) and dystrophic mdx mice (C57BL/10ScSn-DMD^mdx/J, The Jackson Laboratory, Bar Harbor, ME, USA) were purchased from Charles River. Five-month-old wild-type and mdx mice were used for experiments. All experimental protocols and procedures were conducted following the National Ethical Guidelines (Italian Ministry of Health; D.L. 26, 4 March 2014), approved by the local ethics committee (protocol number 375/2019/PR). Animals were housed at controlled temperature (22 ± 1 °C) and humidity (60 ± 5%) and maintained under a 12 h/12 h light/dark cycle with ad libitum access to food and water.
Mice were euthanized and then dissected in order to carefully excise tibialis anterior (TA) muscles from the hind limbs. Collected TA muscles were mounted in Optimal Cutting Temperature (OCT, Tissue Tek^®, Sakura Finetek, Alphen aan den Rijn, The Netherlands, Europe) compound and then frozen in liquid nitrogen-cooled isopentane (2-methylbutane; Sigma-Aldrich, Merck KGaA, Burlington, MA, USA). Embedded muscles were then cross-sectioned at a thickness of 8 µm using a Leica cryostat (Leica CM1850UV, Wetzlar, Germany) set at −25 °C, and sections were stored in a −80 °C freezer.