Immunolabelling was carried out after in situ zymography, as described (Yeghiazaryan et al. 2012 (link)). Goat polyclonal anti-calreticulin, and anti-golgin antibodies were a gift from Dr. Marek Michalak from the University of Alberta (Canada). The anti-calreticulin antibody labels endoplasmic reticulum with high specificity, whereas the anti-golgin antibody detects Golgi structure. To visualize nerves and to analyze their internal architecture, we used antibodies against neurofilament (marker of axons) (Dako, Gdynia, Poland), S100β protein (marker of Schwann cells) (Sigma-Aldrich, Poznan, Poland), or myelin basic protein (MBP, marker of myelin sheath) (Sigma-Aldrich). To label presynaptic structure we used anti-synaptophysin antibody (Dako, Gdynia, Poland). To localize NMJ, the specimens were co-stained with an Alexa 488 or 555 fluorophore-conjugated αBt (Invitrogen/Molecular Probes). To visualize basement membrane, a monoclonal mouse anti-β-dystroglycan was used, 1:10 (Novocastra). The immunoreactions were visualized using species-specific secondary antibodies, coupled with a range of Alexa fluorophores (Alexa 488 or Alexa 555 or Alexa 647, all from Invitrogen/Molecular Probes), diluted 1:200. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) or TO-PRO-3 (Invitrogen/Molecular Probes).
Free full text: Click here
