Zen 2012 blue acquisition software

Sections were examined on a Zeiss LSM5 Pascal (Carl Zeiss Inc.) confocal microscope, a Zeiss LSM 880 NLO, or Leica TCS SP5 (Leica Microsystems, Allendale, NJ, United States) confocal microscope. FITC and Texas Red signals were detected by excitation with the 488 nm and 543 nm lines, respectively. Images were scanned at image scales of 225.0 μm (x) × 225.0 μm (y), 144.72 μm (x) × 144.72 (y), and 450.0 μm (x) × 450.0 μm (y). Captured images were processed using Zen 2012 Blue acquisition software (Carl Zeiss Inc.), Leica Application Suite X software (Version 3.0.2.16120), and Adobe Photoshop CS6 (Adobe Systems Inc., San Jose, CA, United States).

Animals were perfused with 4% paraformaldehyde solution and cryopreserved. Tissues were paraffin-embedded and 20 µm sections were stained with hematoxylin and eosin at the Cancer Mouse Models Core Facility (VCU). For Luxol fast blue staining, paraffin embedded lumbar spinal cord sections were deparaffinized in xylene and stained with Luxol fast blue overnight at 60⁰C. The sections were differentiated in lithium carbonate and 70% ethanol until the gray matter was clear and white matter was sharply defined. Sections were counterstained with hematoxylin. The images were taken on Zeiss Axio imager A1 microscope and processed using Zen 2012 Blue acquisition software (Zeiss Inc.). Quantitative analysis was carried out using Image J software.