Immunohistochemistry and quantification of dystrophin expression. Transverse sections of tissue samples were cut (8-µm thick) for the examination of dystrophin expression. For dystrophin visualisation and quantification, sections were costained with rabbit-anti-dystrophin (Abcam, Cambridge, MA) and rat anti-laminin (Sigma, St Louis, MO), and detected by goat-anti-rabbit immunoglobulin G Alexa 594 and goat-antirat immunoglobulin G 488 secondary antibodies, respectively (Invitrogen, Carlsbad, CA). Images were captured using a Leica DM IRB microscope and Axiovision software (Carl Zeiss, Cambridge, UK). Quantitative immunohistochemistry was performed as previously described. 16, 34 A representative image for each treatment was taken. For quantification, four representative frames of the dystrophin and correlating laminin were taken for each section (n = 3) of the quadriceps, diaphragm and heart for each treatment. Using ImagePro software, 10 regions of interest were randomly placed on the laminin image which was overlaid on the corresponding dystrophin image. The minimum and maximum fluorescence intensity for 120 regions were recorded for each treatment. The intensity difference was calculated for each region to correct for background fluorescence and untreated mdx and treated mdx were normalized to C57BL10. These values were plotted on a scatter graph. The "relative intensity means" were calculated using a multilevel statistics model. Using these values, the percentage recovery score was calculated by implementing the following equation, as described on the TREAT-NMD website (http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/ DMD_M.1.1_001.pdf): (dystrophin recovery of treated mdx mice-dystrophin recovery of untreated mdx mice)/(dystrophin recovery of C57BL10 mice-dystrophin recovery of untreated mdx mice). Staining of dystrophin associated proteins was performed as previously described 29 using a MOM blocking kit (Vector Labs, Burlingame, CA) and α-sarcoglycan and α-dystroglycan (Novocastra, Newcastle-Upon-Tyne, UK) antibodies (1:100 dilution). Neuronal nitric oxide synthase staining was performed using a goat anti-rabbit antibody (Abcam).