Electron microscopy and analysis was performed as described in (13) . Briefly, after region selection on semi-thin sections collected on glass slides and stained with 1% toluidine blue, ultrathin sections were collected on 3mm x 3mm copper grids and examined using a JEOL 1011 transmission electron microscope, and imaged using a MegaView III CCD cooled camera operated with iTEM AnalySIS software (Olympus Soft Imaging Systems). A minimum of six distinct fields of view were captured at 10 000x magnification per animal, and used to count myelinated axons in FIJI/Image J. For this analysis, DHF treated animals were compared to animals that solely received aCSF vehicle treated animals previously published in (13, 14) (link) as DMSO treated animals were not processed for EM analyses. Sectioning, post-staining and EM imaging were all performed at the Centre for Advanced Histology and Microscopy, Peter MacCallum Centre.
Item analysis
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ITEM AnalySIS is a software application developed by Olympus for image analysis and data processing. The core function of ITEM AnalySIS is to provide users with tools for capturing, processing, and analyzing digital images obtained from various microscopy techniques.
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2 protocols using item analysis
1
Ultrastructural Analysis of Myelinated Axons
2
Ultrastructural Analysis of Corpus Callosum
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Semi-thin (0.5–0.1 μm) sections of caudal corpus callosum in a sagittal plane were collected on glass slides and stained with 1% toluidine blue to select region of analysis. Ultrathin (0.1 μm) sections were subsequently collected on 3 × 3 mm copper grids and specimens examined using a JEOL 1001 transmission electron microscope. Images were captured with MegaView III CCD cooled camera operated with iTEM AnalySIS software (Olympus Soft Imaging Systems GmbH). A minimum of six distinct fields of view were imaged at 5,000 or 10,000× magnification for each animal. The proportion of myelinated axons, axon diameter and g-ratio were analyzed manually using FIJI/ImageJ (National Institutes of Health, Bethesda, MD, USA). For g-ratios at least 100 axons from three mice per group were measured. Resin embedding, sectioning and post-staining and EM imaging were performed at the Peter MacCallum Centre for Advanced Histology and Microscopy.
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