Rabbit anti mcp 1

All neutralizing antibodies were intravenously injected into the tail vein every other day after epoB or vehicle administration. The injection doses of neutralizing antibodies are specified according to the published studies, as follows: goat anti-IL-1α (200 μg per rat; Peprotech, Rocky Hill, NJ, USA), goat anti-IL-4 (250 μg per rat; R&D, Minneapolis, MN, USA), mouse anti-M-CSF (750 μg per rat; R&D), rabbit anti-MCP-1 (500 μg per rat; Peprotech), rabbit anti-TNF-α (100 μg/rat; Peprotech), mouse anti-TGF-β (250 μg/rat; Abcam, Cambridge, UK).^{9 (link), 42 (link), 43 (link), 44 (link), 45 (link), 46 (link)} A mix of isotype antibody served as the control. The number and start time of neutralizing antibodies injection was based on results of cytokines antibody array. SCI-epoB injected with M-CSF antibody or MCP-1 antibody from 0 DPE until 7 DPE or killing are referred to as SCI-epoB-M-CSF Ab or SCI-epoB-MCP-1 Ab, respectively. SCI-epoB injected with neutralizing antibody mix (IL-1α, IL-4, M-CSF, MCP-1, TNF-α, and TGF-β) from 0 DPE until 7 DPE or killing is referred to as SCI-epoB-NAM, and SCI-epoB injected with LIX (C–X–C motif chemokine 5) antibody from 3 DPE until 14 DPE or killing is referred to as SCI-epoB-LIX Ab.

Immunohistochemistry and immunoblotting were performed as previously described 4 (link), 34 (link). The following antibodies for immunohistochemistry were used: rabbit anti-GFP (#A6455, Invitrogen), rabbit anti-MCP-1 (#500-P113, PeproTech), mouse anti-TNFα (#ab8348, Abcam), rabbit anti-NeuN (#ab177487, Abcam), rabbit anti-Iba1 (#019-19741, Wako), rabbit anti-Ki67 (#ab15580, Abcam), rat anti-CD68 (#FA-11, Abcam), rabbit anti-Tmem119 (#ab209064, Abcam), rat anti-P2RY12 (#848001, BioLegend), rabbit anti-Synaptotagmin 1 (#105003, Synaptic Systems), mouse anti-MHC-II (#ab55152, Abcam), and mouse anti-PSD-95 (#MAB1596, EMD Millipore). Species-specific secondary antibodies were conjugated with Alexa Fluor488, 594 or 647 (Molecular Probes, Invitrogen). The antibodies for immunoblotting were: rabbit anti-iNOS (#610332, BD PharMingen), mouse anti-COX2 (#610204, BD PharMingen), and mouse anti-GAPDH (#MAB374, EMD Millipore).

Equal amounts of mice spinal cord homogenates protein (40 μg) were resolved separately for soluble and membrane fractions on SDS-PAGE, transferred to nitrocellulose membrane, and blocked overnight with 5 % skim milk in TBS-T (0.3 % Tween 20). Blots of the soluble fraction were probed with the following primary antibodies: mouse anti-actin (1:10,000 Sigma-Aldrich, USA), rabbit anti-MCP-1 (1:1000 Peprotech, USA), rabbit anti-IL-6 (1:1000 Peprotech, USA), and mouse anti-Iba-1 (1:1000 Millipore, Germany). Blots of the membrane fraction were probed with the following primary antibodies: mouse anti-actin (1:10,000 Sigma-Aldrich, USA), rabbit anti-occludin (1:1000 Abcam, UK), rabbit anti-claudin 5 (1:500, Sigma- Aldrich, USA), rabbit anti-ZO-1 (1:1000 Sigma-Aldrich, USA), and rabbit anti-cd36 (1:1000 Abcam, UK). Blots were incubated with corresponding secondary antibodies conjugated peroxidase (Sigma- Aldrich, USA) and developed with the EZ-ECL detection kit (Biological Industries, Israel). Quantitative densitometric analysis was performed using the densitometric software EZQuant-Gel (version 2.12).