Fig. 4.
MABs + mMAG can express and synthesize miniagrin and when injected in dy2J colonize the skeletal muscles but not the peripheral nerves. a C57 MABs transduced with miniagrin gene carrying myc tag show diffuse positivity when stained with anti-myc antibody (a DAPI staining identifies nuclei); scale bar = 30 μm. b Western blot analysis with anti-myc antibody, recognizing mMAG, of cell homogenate and medium from cultures of C57 MABs and C57 MABs transduced with miniagrin gene (MABs + mMAG). Anti-myc recognizes a band only in the homogenate and medium of transduced MABs. β-Tubulin is shown as loading control (accordingly, staining is not present in medium). Each lane is loaded with 25 μg of proteins. b Cryosection of the tibialis anterior muscle from dy2J mouse treated for 10 days with MABs + mMAG. Double staining with anti-laminin γ1 (a depicting myofibers, in red) and anti-myc antibody (b recognizing mMAG, in green) shows diffuse expression of miniagrin around myofibers; scale bar = 50 μm. c Same muscle showing myc-positive fibers with centrally located nuclei (identified by DAPI, in blue), suggesting regenerating fibers; scale bar = 25 μm. c Cryosection of the tibialis anterior muscle from dy2J mouse treated for 10 days with MABs + mMAG. Double staining with anti-laminin γ1 (a depicting myofibers, in red) and anti-myc antibody (b recognizing mMAG, in green) shows that myc positivity is not diffuse to the entire muscle but limited to the area of injection (asterisk). Scale bar = 50 μm. d Cryosection of the tibialis anterior muscle from dy3K mouse 10 days after i.m. injection of MABs carrying a myc tag. Staining with anti-myc antibody recognizes an area where MABs secreted mMAG a, whereas staining with anti-laminin α2 antibody does not show positive signal (b DAPI staining identifies nuclei). Scale bar = 25 μm. e Cryosection of intramuscular nerves of the tibialis anterior muscle from dy2J mouse treated for 10 days with MABs + mMAG. Double staining with neurofilaments (NF) recognizing axons a and anti-myc antibody b recognizing mMAG. Myc staining is restricted to the perineurium and blood vessels, whereas only scarce and scattered positivity is present in the endoneurium. DAPI staining identifies nuclei. Scale bar = 20 μm