To reduce the numbers of mice in our experiments, considering that a similar phenotype was observed in the Tibialis Anterior (TA) and Gastrocnemius (GA) muscles, TA muscles were consistently used for morphological evaluation of the phenotype. Muscles were dissected, embedded in tissue freezing medium (Leica, Wetzlar, Germany) and frozen in liquid nitrogen pre-cooled isopentane (Sigma-Aldrich, Saint Louis, Missouri, # PHR1661). Cryosections (8 μm or 20 μm) were obtained by using a Leica cryostat.
Ventral spinal cords were isolated from perfused mice, embedded in tissue freezing medium (Leica, Wetzlar, Germany) and frozen in liquid nitrogen pre-cooled isopentane (Sigma-Aldrich, Saint Louis, Missouri, # PHR1661). Cryosections (16 μm) were obtained by using a Leica cryostat.
Hematoxylin and eosin (H&E) (Sigma-Aldrich, Saint Louis, Missouri, # H3136 and # 861006) staining was performed according to the Sigma-Aldrich manufacturer's instructions. Cryosections of spinal cords (16 μm) were also stained with NISSL staining (Sigma-Aldrich, Saint Louis, Missouri, #C5042), performed according to the Sigma-Aldrich manufacturer's instructions.
Ventral spinal cords were isolated from perfused mice, embedded in tissue freezing medium (Leica, Wetzlar, Germany) and frozen in liquid nitrogen pre-cooled isopentane (Sigma-Aldrich, Saint Louis, Missouri, # PHR1661). Cryosections (16 μm) were obtained by using a Leica cryostat.
Hematoxylin and eosin (H&E) (Sigma-Aldrich, Saint Louis, Missouri, # H3136 and # 861006) staining was performed according to the Sigma-Aldrich manufacturer's instructions. Cryosections of spinal cords (16 μm) were also stained with NISSL staining (Sigma-Aldrich, Saint Louis, Missouri, #C5042), performed according to the Sigma-Aldrich manufacturer's instructions.