Cd31 ab28364

The tissue sections were transferred directly into antigen retrieval solution (sodium citrate, pH 6.0) and then blocked with a mixture of 3% goat serum, 1% bovine serum albumin (BSA), and 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 1 h at room temperature. Subsequently, the sections (20 μm) were probed overnight with the primary antibodies at 4 °C and then incubated with the secondary antibodies for 1 h at room temperature. The cell nuclei were counterstained with DAPI and the images were recorded using confocal laser scanning microscope (FV3000, Olympus, Tokyo, Japan). The following primary antibodies were used: Endomucin (sc-65495, SANTA CRUZ, Dallas, TX, USA) and CD31 (ab28364, Abcam, Cambridge, UK). The secondary antibodies used included anti-rat Alexa Fluor 488 (4416, Cell Signaling, Boston, MA, USA) and anti-rabbit Alexa Fluor 649 (BS10034, Bioworld, Dallas, TX, USA).

Following mice sacrifice, tumors were fixed in 4% paraformaldehyde. Tissues were dehydrated using alcohol gradient and embedded in paraffin. Five-micrometer sections were taken, deparaffinized, and dehydrated using xylene and alcohol gradients, respectively. The slides were incubated in 10 mM citric acid buffer, pH 6.0 at 100 °C for 20 min for antigen retrieval. The endogenous peroxidase activity was blocked with H₂O₂ (0.3%). Sections were then blocked for 1 h at room temperature with blocking solution (PBS, 0.1% TWEEN, 5% BSA), followed by incubation with primary antibodies. Ki67 (275R-1, Sigma, St. Louis, MO, USA), pS6RP (4857S, Cell Signaling, Danvers, MA, USA), and CD31 (ab28364, Abcam, Cambridge, UK) antibodies were diluted in blocking solution and incubated overnight at 4 °C. The ABC kit (VECTASTAIN Cat. VE-PK-6200) was used for detection according to the manufacturer’s protocol. Sections were counterstained with hematoxylin, dehydrated, and mounted with mounting media (Micromount, Leica, Cat. 380-1730, Wetzlar, Germany). IHC slides were digitalized using the Pannoramic Scanner (3DHISTECH, Budapest, Hungary) and analyzed using QuantCenter (3DHISTECH) using a single threshold parameter for all images of a specific staining sample in each experiment.

The peptide fragment containing amino acids 35–55 of Myelin Oligodendrocyte Glycoprotein (MOG_35–55) was obtained from Sheldon Biotechnology (Montreal, QC, Canada). Complete Freund’s Adjuvant (CFA) containing mycobacterium tuberculosis H37RA (5 mg/ml), pertussis toxin, ovalbumin, horseradish peroxidase (type IV; HRP), decalcifying solution-lite and chemicals used for immunohistochemistry, hematoxylin and eosin staining and luxol fast blue-cresyl violet staining were obtained from Sigma-Aldrich (Oakville, ON, Canada). Immunohistochemistry on mouse spinal cord sections was performed using rabbit anti-mouse polyclonal antibodies from Abcam (Cambridge, MA, USA; product code in brackets): CD31 (ab28364) and vascular endothelial growth factor/VEGF-164 (ab46154). B20-4.1.1 was donated for research use by Genentech Inc. (South San Francisco, CA, USA). K(1-3) (recombinant human angiostatin, kringle domains 1–3) was obtained from Cedarlane (Burlington, ON, Canada). B20-4.1.1 and K(1-3) were stored at 4°C prior to use. Treatment doses were prepared by diluting each agent in sterile PBS at room temperature on the day of injection.