Cryostat machine

All animal procedures were performed at the Translational Cardiomyology laboratory according to the guidelines of the Animal Welfare Committee of KU Leuven (Ethical Committee approval number P161/2018.) and Belgian/European legislation. Mice were sacrificed by cervical dislocation, skeletal muscles and hearts were snap frozen in liquid nitrogen-cooled isopentane and kept at −80 °C for further analyses. The samples were cut transversally in 7 µm sections using a cryostat machine (Leica, Wetzlar, Germany). Immunofluorescence analyses were performed to characterize MICAL2 in skeletal muscle of different murine models and to check MICAL2 protein in AAV-Mical2 H11^Cas9 and in AAV-SHAM H11^Cas9 overtime, not only in skeletal muscle, but also in hearts. Moreover, cryosections were also used for Hematoxylin & Eosin staining and Picro-Sirius red staining on AAV-Mical2 H11^Cas9 and in AAV-SHAM H11^Cas9 skeletal and cardiac muscles.

C57BL6/Cx3cr1-GFP transgenic mice were sacrificed by decapitation. The whole pituitary was quickly dissected and fixed in 1% PFA containing 30% sucrose overnight at 4 ⁰C. The fixed tissue was then washed and equilibrated in half Tissue-Tek O.C.T Compound (Sakura) and half 60% sucrose (final 30%) mixture before positioned inside a plastic mold with only O.C.T compound and frozen by burying in dry ice powder. After the whole block turned opaque, it was stored at –80°C in a sealed plastic bag in the dark. Before cryotomy, the embedded O.C.T block was first equilibrated inside the Cryostat machine (Leica) to –25°C for 30 min followed by cryo-sectioning (7 µm) and slice collection on 22 × 22-mm glass coverslips #1 (Thermo Scientific Menzel), precoated with 0.01% L-lysine (Sigma), and stored at –80°C in a Parafilm sealed six-well plate in the dark for up to a month before further digestion and prehybridization steps. smFISH was conducted as described in (Ji and van Oudenaarden, 2012 (link)) with the exception that the formamide concentration was increased to 30% for prehybridization and washing. Tissue sections were mounted on Prolong Gold antifade mountant (Thermo Fisher) and images were captured using a wide-field fluorescent microscope (Nikon Eclipse Ti-E) with a cooled CCD camera equipped with oil immersion 60× objective.

Intramuscular injections of 2.5 × 10⁵ human MABs were performed in the TA of 3‐month‐old Sgcb‐null Rag2‐null γc‐null mice, available at KU Leuven SPF animal care facility, Belgium (n = 6). MABs from three different donors at P1‐P2 were used. Uninjected limbs were used as controls. After 21 days, mice were sacrificed and muscles were snap‐frozen in liquid nitrogen‐cooled isopentane and then stored at −80°C for further analysis. The samples were then cut transversally in 7‐μm sections using a cryostat machine (Leica, Wetzlar, Germany). Immunofluorescence analyses were performed to study the tissue. Mouse procedures were performed according to the guidelines of the Institutional Animal Care and Use of KU Leuven, under the approved project with protocol code ECD N°P095/2012 issued by the Ethische Commissie Dierproeven of the KU Leuven.