Cresyl violet eosin

Two sets of transverse sections (each set containing serial sections spaced 500 μm apart) from rat spinal cord at 7 weeks post-SCI were stained with Luxol Fast Blue (LFB; Sigma) for white matter sparing analysis (n = 10/group) and cresyl violet-eosin (Sigma) for lesion volume assessment (n = 10/group), as previously described (Hu et al., 2013 (link)). White matter sparing was defined as tissue exhibiting normal myelin appearance and density; the lesion center was defined as the section containing the least amount of spared white matter. The areas of whole transverse sections and myelinated white matter at the lesion center were outlined and quantified in LFB-stained sections, and are expressed as a percentage of the total stained area. After cresyl violet-eosin staining, total and cross-sectional areas of the spinal cord and lesion boundary were measured using the Neurolucida System (MicroBrightField, Colchester, VT, USA) connected to a BX60 microscope (Olympus, Tokyo, Japan). The total volume of the lesion area (including areas of cavitation and degeneration) was calculated as the sum of the individual sub-volumes, which were determined by multiplying the cross-sectional area by the distance between sections (500 μm). The percent total volume of the injured area was calculated by dividing the total volume of the lesion area by that of the corresponding length of spinal cord.

Two sets of transverse sections (each set containing serial sections spaced 500 μm apart) from rat spinal cord at 6 weeks post-SCI were stained with cresyl violet-eosin (Sigma) for lesion area assessment (n = 8/group) and Luxol Fast Blue (LFB; Sigma) for white matter sparing analysis (n = 8/group), as previously described (Hu et al., 2013 (link)). The total area of cavitation and LFB-positive myelinated areas in axial sections at the injury epicenter and at 1, 2, and 4 mm rostral and caudal to the injury epicenter were outlined and measured using the Neurolucida System (MicroBrightField, Colchester, VT, United States) connected to a BX60 microscope (Olympus, Tokyo, Japan), and are expressed as a percentage of the total stained area. The lesion center was defined as the section containing the least amount of spared white matter.

Longitudinal sections (20 µm; 500-μm intervals, 8 sections per animal; n = 10) of fixed, frozen mice spinal cord tissue were stained at 8 weeks post-SCI. They were stained with Cresyl Violet eosin (Sigma-Aldrich Ltd., Rehovot, Israel) for lesion area assessment and Luxol Fast Blue (LFB; Sigma-Aldrich Ltd., Rehovot, Israel) for white matter sparing analysis. The center of each lesion was defined as the section containing the least amount of spared white matter. LFB-positive myelinated areas were measured at the epicentre, and different distances, rostral and caudal from the epicentre, were recorded as specified.
Sections were imaged by fluorescence microscopy using an Olympus IX83 fluorescence microscope, an ORCA digital camera, and cellSens Dimension Version 3; images were sized using Adobe Photoshop 11 and Illustrator 14. All fluorescence density or intensity measurements were performed using ImageJ software.