M1 compound

Sections of the spinal cord and brain were analyzed by hematoxylin and eosin (H&E) and immunohistochemical staining. The rats were perfused with 0.9% normal saline, followed by 4% paraformaldehyde (Hushi Inc., Shanghai, China) in 0.1 M PBS (pH 7.4). The brains and spinal cords were dissected from the rats, postfixed overnight in 4% paraformaldehyde at 4 °C, and cryopreserved with 30% sucrose for 3 days. The tissue samples were embedded in the M1 compound (Thermo Fisher Scientific Inc., Waltham, MA, USA), the spinal cords were cut into 16 µm sagittal sections, and the brain samples were cut into 10 µm coronal sections using a cryotome cryostat. H&E staining was performed on the lesion epicenter to examine the general morphology of the spinal cord at 12 weeks after injury, and three sagittal sections containing a lesion cavity and four cases per group were evaluated for quantitative analysis. The size of the lesion cavity was manually outlined for each section under confocal microscopy and analyzed using Image J software (1.37 v, National Institutes of Health, Bethesda, MD, USA) as our previous studies [28 (link),29 (link)].

For histological analysis, all animals were deeply anesthetized and transcardially perfused with 150 mL of saline, followed by 500 mL of 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.2) via a peristaltic pump. After perfusion, the spinal cord was removed, postfixed in 4% PFA overnight, and cryoprotected in 30% sucrose for 3 days. The tissues were embedded in M1 compound (Thermo Fisher Scientific Inc., Waltham, MA, USA) and sectioned sagittally at a thickness of 16 µm on a cryostat. H&E staining was performed at each time point after SCI. Sections were washed in 0.1 M PBS and immersed in Harris hematoxylin solution (Merck KGaA, Darmstadt, Germany) for 3 min followed by a brief wash in distilled water. Slides were then immersed briefly in 1% acid alcohol (1% HCl in 70% ethanol) and stained with eosin Y solution (BBC biochemical, Mount Vernon, WA, USA) for 30 s. The slides were dehydrated with an ethanol series, cleared with xylene, mounted in DPX (Merck KGaA, Darmstadt, Germany), and observed under a microscope (Nikon, Tokyo, Japan).