Luxol fast blue (lfb)

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.

The remaining rats were anesthetized with 10% trichloroacetaldehyde at 6 weeks post-SCI, and cardiac perfusion was performed with PBS followed by precooled PFA at 4°C. Spinal cords were collected, fixed, and cut into 10 μm serial frozen sections using a frozen microtome (Leica), as described in the immunofluorescence protocol. According to the manufacturer’s instructions, the slides were stained with the desired staining. Hematoxylin-eosin (HE, Beyotime) and Luxol Fast Blue (LFB, Sigma-Aldrich) staining were performed to measure the lesion area and the preservation of myelination (LFB-positive area) of the spinal cord. Lesion and myelinated area measurements were performed in an unbiased stereological manner using ImageJ software. Cavitation and LFB-positive tissue at the injury epicenter and the epicenter to rostral and caudal 1, 2, 3, and 4 mm in axial sections were quantified and normalized to the percentage of intact spinal cord area. Nissl staining (Beyotime) was used to identify surviving motor neurons by the existence of the Nissl substance and euchromatic nuclei. Surviving motor neurons were quantified by counting all such cells in the ventral horn. The three staining methods were described previously (Chen et al., 2020 (link)).

The rats were euthanized on the 28th day after operation. Tissue samples were randomly cut and soaked in 4% paraformaldehyde for 48 h and then embedded in paraffin. Nerve sections were deparaffinized in xylene (2 × 5 min), hydrated in graded ethanol (2 × 5 min in 100%, 5 min in 85%, and 5 min 70%) followed by distilled water and finally rinsed in PBS. Next, the sections were stained overnight at 56 °C in 0.1% Luxol fast blue (LFB) (Sigma, St. Louis, MO, USA) in acidified 95% ethanol, rinsed in 95% ethanol and differentiated in 95% ethanol. For the detection of the percentage of myelin area in rats, we randomly selected three rats from each group, and each rat made two sections for Luxol Fast Bule staining and scanning. The scanned images were enlarged to 50 μm by Caseviewer software and then five faces were randomly selected. The percentage of myelin area of rats was calculated, Image-Pro Plus software was used to calculate and the histogram was made by PRISM software.

On the week four, rats were humanely euthanatized and nerves immersed in 4% paraformaldehyde for 48 h and paraffin-embedded. The sections of the nerve were deparaffinized in xylene and hydrated in 100% ethanol, 85% ethanol, and 85% ethanol for 5 min each and finally rinsed in PBS. Next, the sections were stained overnight at 56°C in 0.1% Luxol Fast Blue (LFB) (Sigma, St. Louis, MO, USA) in acidified 95% ethanol and then rinsed in 95% ethanol and differentiated in 95% ethanol.