Metamorph software

To evaluate the migratory characteristics of NSCs in vitro, we used a similar system described previously ^{29 (link)}, with slight modifications. BD Biocoat Tumor Invasion System containing BD Falcon Fluoroblock 24-Multiwell inserts (8-μm pore size; PET membrane) was used following the manufacturer’s instructions (BD Biosciences, Bedford, MA, USA). The migration of GFP-labeled NSCs was characterized towards conditioned media obtained by culturing 5 × 10⁴ GBM43 cells in serum-free/growth factor-free media for 24 hours. The medium was added to the bottom well of the migration chamber to act as a chemoattractant. NSCs were then plated in the top insert at a density of 3 × 10⁵ cells per well in DMEM (Mediatech, Manassan, VA) along with 5 × 10⁴ glioma cells in the lower chamber of the 24-well plate; the cells were kept in these conditions for 48 hours. Cells located above the migration filter were classified as non-migratory and those just below the filter as migratory; each group of cells was collected by using cell scrapers for subsequent gene expression, qRT-PCR, and flow cytometry analysis. The number of migrating cells in three random fields of views per well was counted using an Olympus IX81 inverted microscope (original objective: 20X) and analyzed using the MetaMorph software (Olympus, Tokyo, Japan).

EDL and soleus muscles were dissected and freed from surrounding tissues, the tendons pinned at the bottom of a plastic histology mold, the muscle cryopreserved in 5% sucrose overnight, followed by 20% sucrose overnight, then washed in PBS and covered with optimal cutting temperature compound (O.C.T.), and frozen at −80 °C until used. In preliminary experiments, we exposed muscles sections to a buffer containing the calcium chelator 3% ethylenediaminetetraacetic acid (EDTA) in PBS to prevent contraction; however, they did not show significant differences in the muscle and NMJ organization compared to those in control conditions devoid of EDTA. Muscles were longitudinally sectioned (25 μm) with a Leica CM3050S cryostat, mounted on glass slides, fixed in 2% PFA for 30 min at room temperature, washed in PBST, then in PBS, incubated in 1:250 in PBS tetramethylrhodamine 554-α-bungarotoxin-conjugate for 3 h, rinsed in PBS, and mounted using Dako mounting medium. The preparation was imaged using an Olympus FV1200 spectral laser scanning confocal microscope, an UPLSAPO20X NA: 0.75 objective, and Olympus MetaMorph software, at 2 μs/pixel, and 0.31 μm/pixel.