Rhodamine conjugated α bungarotoxin

Anti-ganglioside GM1 antibody was purchased from Millipore (345757). Rabbit polyclonal antibody to Galgt1 peptide CQVRAVDLTKAFDAEE was made in our lab by immunizing rabbits with KLH-conjugated peptide, after which antibody was purified over peptide-conjugated resin as previously described [61 (link)]. Anti-mouse Pax7 antibody was a gift from Dr. Michael Rudnicki (Ottawa Health Research Institute). Anti-mouse integrin α7 conjugated to fluorescein isothiocyanate (FITC) was purchased from MBL International (K0046-4) and R & D Systems (FAB3518F). Anti-mouse CD11b conjugated to FITC and Rat anti-Ertr7 were gifts from Dr. Jill Rafael-Fortney (The Ohio State University). Rat anti-mouse Ly-6A/E conjugated to FITC (Sca1, 553335), rat anti-mouse CD45 conjugated to PE-Cy7 (552848), rat anti-mouse CD31 conjugated to APC (551262), and rat anti-mouse CD16/CD32 Fc block (553142) were purchased from BD Biosciences. All secondary antibodies conjugated to fluorophores were purchased from Jackson ImmunoResearch. Rhodamine-conjugated α-bungarotoxin was purchased from Life Technologies. Sections from normal human and Duchenne muscular dystrophy muscle biopsies from clinical specimens archived as part of the United Dystrophinopathy Project were obtained in accordance with approval from the Institutional Review Board.

Muscle re-innervation was also assessed by counting the number of re-innervated motor endplates. The number of presynaptic terminals immunoreactive to the synaptic vesicle protein, SV2, was assessed three and four weeks following nerve transection and repair. The re-innervated gastrocnemius muscles were harvested and cryoprotected in sucrose, as described above. Muscles were then sectioned on a cryostat in a horizontal plane at 20 µm thickness, placed on slides, and reacted with an antibody to SV2 (1:50; Developmental Studies Hybridoma Bank) for 72 hours at 4° C, followed by a goat anti-mouse immunoglobulin secondary antibody conjugated to Alexafluor 647 (1:200; Life Technologies D22914), and rhodamine-conjugated α-bungarotoxin (1:1000; Life Technologies B35451). In each muscle studied (20 cases), fluorescent images of 50 motor endplates marked by bungarotoxin binding were obtained and scored for immunoreactivity to SV2 within the boundaries of the endplate, indicating the presence of an overlying neuromuscular synapse. The percentage of re-innervated (SV2+) gastrocnemius motor endplates was compared between treatment groups (untreated and optically treated, each as a percentage of intact).