Nuclei were immunostained with P7 dystrophin antibody [41] and counterstained with 49,6-diamidino2-phenylindole (DAPI) fluorescent dye (Sigma, UK). The expression of myosin 3F-nLacZ-2E by dystrophin-positive fibres is evidence that the group of fibres was of donor origin [6,7], rather than being host (revertant) [42,43] fibres. Quantification of donorderived nuclei and fibres was performed in the section with the highest number of donor-derived dystrophin-positive fibres [6,7]. Analyses of muscle cross section area (CSA), number and myofibre area were performed on 11967625 cryo-sections that had been stained with polyclonal laminin antibody (Sigma, UK) or with haematoxylin and eosin (H E) [44]. Serial transverse sections were cut throughout the entire muscle and the largest transverse section was selected for analysis. Multiple images, captured at 106 magnification, from the selected section were assembled to give an image of the entire section and this was used for quantification of CSA and number and area of myofibres.Donor Mouse ModelsAdult (2? months old) genetically modified 3F-nlacZ-2E and bactin-Cre:R26NZG (obtained from crossing a homozygote male b-actin-Cre (FVB/N-Tg(ACTB-cre)2Mrt/J) -a kind gift from Massimo Signore, UCL- with an homozygote female R26NZG (Gt(ROSA)26Sortm1(CAG-lacZ,-EGFP)Glh) (The Jackson Laboratory, USA)) mice were used as donors. b-galactosidase (b-gal) is expressed in all myonuclei in 3F-nlacZ-2E mice [34] and ubiquitously in all nuclei of b-actin-Cre:R26NZG mice [35,36]. These two models allow us to identify either myonuclei alone, or all nuclei (including those outside myofibres) of donor origin, within grafted muscles.Image Capture and Quantitative AnalysesFluorescence and brightfield images were captured using a Zeiss Axiophoto microscope (Carl Zeiss, UK) and MetaMorph image capture software (MetaMorph software, USA). Digitalization of images and quantification were performed with ImageJ (rsbweb.nih.gov/ij). Graph and figures were assembled using Photoshop CS2 software.Statistical AnalysesResults are reported as mean 6 SEM from an appropriate number of samples, as order CUDC-907 detailed in the figure legends. Student’s ttest and Chi-squared test were performed using GraphPad software to determine statistical significance.Donor Fibre and Satellite Cell PreparationExtensor digitorum longus (EDL) muscles were isolated from donor mice as previously described [37,38]. Briefly, after mice were killed by MedChemExpress CTX-0294885 cervical dislocation, EDL muscles were carefully isolated from tendon to tendon under microscopic observation and digested in 2 collagenase type I (Sigma)/DMEM at 35uC for 70 minutes. Muscle fibres could then be easily separated under a stereo microscope by using heat-polished, pulled glass Pasteur pipettes. Fibres were serially washed to eliminate debris and other muscle components and only intact, clean myofibres were carefully selected. Some fibres were carefully transferred in a plate in DMEM and kept at 37uC for less than an hour before 4 ml of DMEM containing one fibre were grafted into the middle part of each host muscle by means of fine glass needle. In the experiments where satellite cells, rather than isolated fibres, were grafted, an aliquot of the fibre preparation was triturated to release satellite cells [7,38,39] and approximately 4 ml of DMEM-containing 400 of these cells was grafted into TA muscles of host mice by means of fine glass needle [7,40]. Host mice were grafted 3 days after muscle injury and muscles were r.Nuclei were immunostained with P7 dystrophin antibody [41] and counterstained with 49,6-diamidino2-phenylindole (DAPI) fluorescent dye (Sigma, UK). The expression of myosin 3F-nLacZ-2E by dystrophin-positive fibres is evidence that the group of fibres was of donor origin [6,7], rather than being host (revertant) [42,43] fibres. Quantification of donorderived nuclei and fibres was performed in the section with the highest number of donor-derived dystrophin-positive fibres [6,7]. Analyses of muscle cross section area (CSA), number and myofibre area were performed on 11967625 cryo-sections that had been stained with polyclonal laminin antibody (Sigma, UK) or with haematoxylin and eosin (H E) [44]. Serial transverse sections were cut throughout the entire muscle and the largest transverse section was selected for analysis. Multiple images, captured at 106 magnification, from the selected section were assembled to give an image of the entire section and this was used for quantification of CSA and number and area of myofibres.Donor Mouse ModelsAdult (2? months old) genetically modified 3F-nlacZ-2E and bactin-Cre:R26NZG (obtained from crossing a homozygote male b-actin-Cre (FVB/N-Tg(ACTB-cre)2Mrt/J) -a kind gift from Massimo Signore, UCL- with an homozygote female R26NZG (Gt(ROSA)26Sortm1(CAG-lacZ,-EGFP)Glh) (The Jackson Laboratory, USA)) mice were used as donors. b-galactosidase (b-gal) is expressed in all myonuclei in 3F-nlacZ-2E mice [34] and ubiquitously in all nuclei of b-actin-Cre:R26NZG mice [35,36]. These two models allow us to identify either myonuclei alone, or all nuclei (including those outside myofibres) of donor origin, within grafted muscles.Image Capture and Quantitative AnalysesFluorescence and brightfield images were captured using a Zeiss Axiophoto microscope (Carl Zeiss, UK) and MetaMorph image capture software (MetaMorph software, USA). Digitalization of images and quantification were performed with ImageJ (rsbweb.nih.gov/ij). Graph and figures were assembled using Photoshop CS2 software.Statistical AnalysesResults are reported as mean 6 SEM from an appropriate number of samples, as detailed in the figure legends. Student’s ttest and Chi-squared test were performed using GraphPad software to determine statistical significance.Donor Fibre and Satellite Cell PreparationExtensor digitorum longus (EDL) muscles were isolated from donor mice as previously described [37,38]. Briefly, after mice were killed by cervical dislocation, EDL muscles were carefully isolated from tendon to tendon under microscopic observation and digested in 2 collagenase type I (Sigma)/DMEM at 35uC for 70 minutes. Muscle fibres could then be easily separated under a stereo microscope by using heat-polished, pulled glass Pasteur pipettes. Fibres were serially washed to eliminate debris and other muscle components and only intact, clean myofibres were carefully selected. Some fibres were carefully transferred in a plate in DMEM and kept at 37uC for less than an hour before 4 ml of DMEM containing one fibre were grafted into the middle part of each host muscle by means of fine glass needle. In the experiments where satellite cells, rather than isolated fibres, were grafted, an aliquot of the fibre preparation was triturated to release satellite cells [7,38,39] and approximately 4 ml of DMEM-containing 400 of these cells was grafted into TA muscles of host mice by means of fine glass needle [7,40]. Host mice were grafted 3 days after muscle injury and muscles were r.
