**2. Materials and Methods**

### *2.1. Mouse Strains and Animal Procedures*

C57BL/6J (RRID:IMSR\_JAX:000664) and C57BL/10ScSn-Dmdmdx/J mice (RRID:IMSR\_JAX:001801), hereafter referred to as wt and mdx mice, respectively, were purchased from the Jackson Laboratory.

Mice were bred respecting the standard animal facility procedures, and all the procedures were conducted in accordance with rules of good animal experimentation I.A.C.U.C. n◦432 of 12 March 2006 and under ethical approval released on 23/October/2017 from the Italian Ministry of Health, protocol #820/2017-PR.

For muscle injury, 45-day-old wt and mdx mice were anesthetized with an intramuscular injection of saline solution containing ketamine (5 mg/mL) and xylazine (1 mg/mL) prior to the intramuscular administration of 20 μL of 10 μM cardiotoxin solution, isolated from *Naja Pallida* (Latoxan L8102, Portes les valence, France), into *tibialis anterior*, *quadriceps,* and *gastrocnemius* muscles.

#### *2.2. Histological Analysis*

*Tibialis anterior* (TA) muscles were collected, embedded in optimal cutting temperature compound (Killik—O.C.T., Bio Optica, Milan, Italy), and snap-frozen in liquid nitrogen for 10 s. Embedded muscles were stored at −80 ◦C for transverse cryo-sectioning with a Leica cryostat. Cryosections (10 μm thickness) were collected on Superfrost glass slides (Thermo Fisher Scientific, Monza, Italy), and tissue slides were stained with hematoxylin and eosin (H&E).

For the H&E, cryosections were fixed with 4% paraformaldehyde (PFA, Santa Cruz Biotechnology, D.B.A. Italia S.r.l., Segrate Milan, Italy) for 15 min at room temperature (RT). After washing in 1X PBS, tissue slides were incubated in the hematoxylin solution for 15 min and rinsed for 5 min in tap water. Cryosections were then counterstained with an alcoholic solution of eosin for 30 min. Following the eosin staining, cryosections were dehydrated in increasing concentrations of alcohol, clarified with the histo-clear solution (Agar Scientific Ltd, Stansted, UK), and finally mounted on coverslips, using the resinous Eukitt mounting medium (Electron Microscopy Sciences, Hatfield Township, PA, USA).

H&E images were captured using the Zeiss Lab A1 AX10 microscope at the 20× magnification in the bright field.
