Computational protocol: Paranode Abnormalities and Oxidative Stress in Optic Nerve Vulnerable to Secondary Degeneration: Modulation by 670 nm Light Treatment

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Protocol publication

[…] Longitudinal ON sections were visualised and photographed using a Nikon Eclipse Ti inverted microscope (Nikon Corporation) at emission wavelengths of 420 nm, 590 nm and 515 nm, with a 20X or 40X/1.3 N.A. oil immersion objective. For each animal, a series of optical images at 0.5 µm increments along the z axis were acquired from the middle 6 µm of a single 14 µm thick section of ON at the injury site, sampling a field of view of 217.5×162.5 µm encompassing the ventral half of the ON (vulnerable to secondary degeneration). All images were collected using Nikon Elements AR software and deconvolved using autoquant blind deconvolution. Representative images of node/paranode complexes were taken with a Leica TCS SP2 AOBS Multiphoton Confocal microscope (Leica Microsystems) at emission wavelengths of 519 nm and 565 nm. Images were collected in 0.5 μm optical z-series sections and reconstructed into a single image with Leica imaging software. For imaging of cytochrome c oxidase activity and associated cellular immunohistochemistry, images encompassing the ventral half of the ON were captured using an Olympus Bx50 upright microscope for non-fluorescent sections and a Leitz Laborlux S upright microscope for fluorescent sections. [...] Each image stack was opened in NIH ImageJ software using the ImageJ ND2 reader plugin and each image along the z axis was converted to a TIFF file. For analyses where colocalisation of 2 markers was assessed, a single image in the z plane with the strongest staining at all wavelengths was used to ensure optimal visualisation and true colocalisation. The intensity of immunoreactivity of oxidative stress indicators (CML, DHE) in individual cell types (CC1+ve, olig1+ve) was determined by tracing around identified oligodendrocyte lineage cells using the polygon tool and collecting intensity measurements from the same area on the DHE or CML image, with outcome measures of maximum or mean intensity. Note that images within experiments were captured at constant exposures and in a single session, but values obtained cannot be compared to values obtained from different experiments where image exposure times would be different. […]

Pipeline specifications

Software tools AutoQuant, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Retinitis, Wounds and Injuries, Chromosome Disorders
Chemicals Superoxides