Immunohistochemistry

Spinal cords were isolated from rats perfused with 250 ml of chilled PBS followed by 50 ml of chilled 4% paraformaldehyde (PFA). Spinal cords were then postfixed in 4% PFA overnight at 4°C before transfer to 30% sucrose (at 4°C) and storage in octamer‐binding transcription factor (OCT) (at 4°C for 1–2 days then −20°C). Embedding was done in fresh OCT and 20 μm thick cross‐sections were generated.

Slides were baked for 15 minutes at 55°C so cryosections would adhere permanently to the slides. After blocking in PBS + 2% FBS + 0.1% Triton X‐100 for 1 hour at room temperature, primary antibodies diluted in the same diluent solution were applied overnight at 4°C. After three washes in PBS, secondary antibodies (1:200 dilution) were applied as necessary. Hoechst 33242 was used as a nuclear counterstain. Slides were mounted in Mowiol after five PBS washes. Negative staining controls (primary or secondary antibody alone) were used to assess baseline image acquisition parameters. The latter were kept consistent for each fluorescent channel between slides and treatment conditions.

To assess vascularity, endothelial cells were labeled with a DyLight 594‐conjugated tomato lectin from Lycopersicon esculentum agglutinin (LEA, DL‐1177, VectorLabs, Canada, 1:300). Myelination and axonal density were quantified using fluro‐myelin ({"type":"entrez-nucleotide","attrs":{"text":"F34651","term_id":"4820277","term_text":"F34651"}}F34651, Molecular Probes; Eugene, OR, http://probes.invitrogen.com, 1:100) and anti‐NF200 (N0142, Sigma–Aldrich, 1:200), respectively. Astrogliosis and glial scarring were quantified using anti‐Glial fibrillary acidic protein (GFAP) (AB5541, Millipore; Burlington, MA, http://www.millipore.com, 1:200) and anti‐Chondroitin sulfate proteoglycan (CSPG) (Clone CS‐56, C8035, Sigma, Canada, 1:200) antibodies, respectively. All appropriate goat secondary antibodies (Alexa Fluor) were used at 1:200 dilution. Unbiased estimation of spinal cord diameter, tissue sparing, and gray‐white matter ratio was carried out on StereoInvestigator software (MBF Bioscience; Williston, VT, https://www.mbfbioscience.com/) on a Nikon Eclipse E800 microscope for longitudinal cryosection slides.