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Revertant fibers were assessed for dystrophin expression by incubation for 1?h at room temperature in an antibody to dystrophin (1:500; abcam). Incubation in primary antibody was followed by a rinse in PBS, followed by sequential incubation in reagents from the peroxidase ABC VectaElite or ABC Vectastain kits (Vector Selleckchem JNJ26481585 Laboratories), and developed with diaminobenzidine containing heavy metals. Additional sections were prepared for the visualization of utrophin (1:200; Santa Cruz, Santa Cruz, CA). The primary antibody to utrophin had an overnight incubation in humid chambers, the slides were rinsed in PBS, and incubated in goat anti-rabbit AF488 secondary antibody (1:2000; Jackson Immunoresearch, West Grove, PA) for 1?h. After a rinse in PBS, the slides were coverslipped with Vectashield mounting medium. Morphometric analysis Morphometric analysis of fiber sizes and myogenic precursor cell density were performed using Bioquant Life Science software using our published methods (Anderson et?al. 2006) (Bioquant, Nashville, TN). A minimum of three slides were counted for each set of triceps muscles examined from each animal, with a minimum of 200 myofibers assayed per slide. Care was taken in all sections (-)-p-Bromotetramisole Oxalate to measure in different regions. For the MyHC isoforms, due to fiber type grouping and heterogeneous localization in the dystrophic samples, 400 fibers were counted per slide to ensure that an adequate area was covered. For the dystrophic muscles, this is less of an issue, due to the changes in fascicular patterns PI3K inhibitor in different regions of the muscle length. Values for each animal were averaged, and the averages for each genotype and age were averaged. For central nucleation, data are presented as percent of centrally nucleated myofibers per total fiber number. For Pax7-positive cell density, data are presented as percent of Pax7-postive cells per total fiber number. Fibrosis was quantified by setting a threshold for the collagen I or IV immunostained tissue, calculating the area of positive immunostaining and dividing this area by total area/sections counted. This gives data as percent fibrosis per total muscle cross-sectional area. Statistics All statistical analyses were performed using Prism statistical software (GraphPad Software Inc., San Diego, CA). Analysis of variance (ANOVA) and Tukey's multiple comparisons tests were used for multiple group comparisons. Data were considered statistically significantly different if P?
