If successful, such strategy would be highly valuable given the scarcity of treatment options for neuromuscular disorders

If successful, such strategy would be highly valuable given the scarcity of treatment options for neuromuscular disorders. Conclusion The current study shows that in panels). supplementary material, which is available to authorized users. gene [6]. We performed homozygous breedings to generate both the wildtype and animals in the FVB/N background during the duration of this project. Wildtype control and animals in the mixed C57/Bl6 and 129/Sv background were obtained as littermates from heterozygous breedings and maintained at the Cardarelli Hospital s Animal Facility (Naples, Italy). Gaa?/?(Bl6) animals obtained by insertion of a neo cassette into exon 6 of the gene [35] were purchased from Charles River Laboratories (Wilmington, MA). All mice in experiment ITD-1 were housed under a lightCdark cycle (12?h) and under defined pathogen-free conditions, with access to food and water ad libitum. Muscle injury was induced by intramuscular injection of 1.2% (in PBS) BaCl2 or cardiotoxin (CTX; 10?mol in PBS). Animals were allowed to recover for the time indicated in the figures. Serial injury experiments were performed by injecting BaCl2, as described above, three times at monthly intervals into the Tibialis Anterior (TA) muscle. Three weeks after the last BaCl2 injection the animals were sacrificed for tissue collect. At the end of experiments animals were sacrificed by cervical dislocation during daytime without a fixed timepoint. Tissue wet weight was determined by weighing freshly dissected tissue that was blotted dry. All animal experiments were approved by the local and national animal experiment authorities in compliance with the European Community Council Directive guidelines (EU Directive 86/609), regarding the protection of animals used for experimental purposes, and according to Institutional Animal Care and Use Committee (IACUC) guidelines for the care and use of animals in research. The study was approved by the local and national authorities in the Netherlands ITD-1 and Italy, respectively. All procedures with the animals were performed with the aim of ensuring that discomfort, distress, pain, and injury would be minimal. Determination of glycogen levels To measure tissue glycogen concentrations 20 30?m cryosections were collected for each sample. The sections were homogenized using 5?mm stainless steel beads (Qiagen NV) in the Qiagen Retsch MM300 TissueLyser (Qiagen NV) at 30?Hz for 5?min. Glycogen was quantified in tissue supernatant by measuring the amount of glucose released from glycogen after conversion by amyloglycosidase and amylase (Roche Diagnostics) for 1?h as previously described [58]. Spectral absorbance of the products was measured on a Varioskan spectrometer (Thermo Scientific) at 414?nm. Results from ITD-1 the glycogen measurements were normalized for protein content using the Pierce BCA protein assay kit (Thermo Scientific). Histology and immunofluorescent analyses Hematoxylin and Eosin (HE) staining and Massons trichrome staining CR1 were performed using routine histology protocols as described previously [41]. For immunostaining, Tissue-Tek OCT-embedded tissue was snap-frozen in liquid nitrogen-cooled isopentane. 10?m cryosections were cut and fixated in ice-cold aceton. A heated antigen retrieval procedure with 10?mM citrate buffer was used for the detection of Pax7. Sections were stained essentially as described previously [41], but using the M.O.M. kit from Vector laboratories for blocking endogenous mouse immunogens. Primary antibodies used were eMyHC (F1.652; DSHB; 1:300), Ki67 (Ab15580; Abcam; 1:50), laminin (L9393; Sigma; 1:500 or LS-C (6142; LS BIO; 1:500)), Lamp1 (Ab24170; Abcam; 1:150). Hoechst ({“type”:”entrez-nucleotide”,”attrs”:{“text”:”H33258″,”term_id”:”978675″,”term_text”:”H33258″}}H33258, Sigma) was used at 1?g/ml. To detect centrally nucleated myofibers aceton-fixed 10?m cryosections were stained for laminin using a primary antibody and Hoechst for nuclei, as described above, and imaged by fluorescent microscopy. Image acquisition and analysis Histological sections were scanned with 4x and 20x objectives on a Hamamatsu NanoZoomer 2.0 (Hamamatsu Photonics). Images were analyzed using NDP view software (NDP View 1.2.31 Eng, Hamamatsu Photonics). Sections used for immunofluorescence were scanned on Zeiss LSM700 (Carl Zeiss B.V.) using tile-scan modality with a 20x objective. Image analysis and processing was performed using Fiji (fiji.sc/Fiji) and Adobe Photoshop. Quantification of myofiber diameter was performed using cross sections by measuring the longest diagonal (in m) in at least 100 fibers per sample, randomly selected throughout the whole section. Flow cytometry Preparation of limb muscle for flow cytometric analysis was adapted from Liu et al. [28]. In short, dissected tissue was minced thoroughly to small pieces in F10 medium (Lonza) containing collagenase II (750?U/ml;.