Sc 304

5–10 μm cryosections of patient and control biopsies were stored until analysis at −80 °C. Sections were thawed before staining. Primary antibodies were directed against CD68, clone KP1 (M0814; DAKO; 1:800), eMyHC (F1.652; DSHB; 1:150), Ki67 (Ab15580; Abcam; 1:50), Laminin (L9393; Sigma; 1:500 or LS-C96142; LS Bio; 1:500), MyoD (SC304; Santa Cruz; 1:200), Myogenin (M-225 ; Santa Cruz; 1:200), Pax7 (DSHB; 1:50), CD3 (2GV6; Ventana; ready to use), CD20Cy; clone L26; Dako, 1:400). Following the primary antibody incubation, sections were rinsed and incubated with biotin-conjugated anti-mouse antibody (BA-2000; Vector labs,1:50) and then with Alexafluor594-conjugated streptavidin (S11227; Life Technologies, 1:500). Sections incubated with rabbit primary antibodies were subsequently incubated with Alexafluor 488-conjugated goat-anti rabbit antibodies (A11307; Life Technologies,1:500). Finally chicken anti-Laminin was detected with Alexafluor647-conjugated goat anti-chicken antibodies (A21449; Life Technologies, 1:500). All sections were counterstained with Hoechst33258 (H3569; Life Technologies, 1:15000). The slides were mounted with Mowiol (475904; Calbiochem). For some biopsies, limited amounts of sections of sufficient quality were available for immunofluorescent analysis, and not all stainings could be performed for all patients.

For western blot analysis, 100 mg of muscle was collected, physically disrupted and homogenized with a pestle in the presence of silicon dioxide powder and 1 ml of lysis buffer [75 mM Tris-HCl (pH 6.8), 10% SDS, 20% glycerol, 5% mercaptan, 0.001% bromophenol blue] and incubated at 95°C for 10 min to denature proteins. Each well on a precast mini-protean TGX 4-15% gel (Bio-Rad) was loaded with 5 μg of total protein. Proteins were transferred onto a nitrocellulose membrane using a standard wet transfer in Mini Trans-Blot Cell (Bio-Rad) using the manufacturer's protocol. Nonspecific binding was blocked by incubating the membranes in 5% dry milk in PBS for 3 h. Antibodies against target proteins (ab154168 1:1000, ab15277 1:400, ab188873 1:1000, ab189254 1:1000, ab15200 1:200, Abcam; NCL-b-DG 1:200, Novocastra; sc-14176 1:200, sc-304 1:200, Santa Cruz Biotechnology) or a rabbit monoclonal anti-actin antibody (A2103 1:10,000, Sigma-Aldrich) diluted in 1% dry milk in PBS were used for protein detection. Incubation with each antibody was carried out overnight at 4°C, followed by washes and incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1/3000) for 1 h at room temperature and further washes in PBS. Enhanced chemiluminescence was used for the detection of protein bands, and images were obtained and analyzed on a C-DiGit Blot Scanner.