The animals were reanesthetized and transcardially fixed with 4% paraformaldehyde. The spinal cord was removed, dehydrated, and embedded in an optimal cutting temperature compound, and frozen sections were cut on a cryostat in the sagittal plane at 14 mm. The sections were subsequently stained with primary antibodies at 4°C overnight and then incubated with Alexa Fluor secondary antibodies (1:200; Invitrogen) and Hoechst 33258. LFB-stained serial sections were evaluated as previously described (4, 9, 15). The R.T.U. (Ready-to-Use) Vectastain kit (Vector Laboratories) was used for CD68 staining (15). The following primary antibodies were used: CD68 (rat, MCA1957; 1:200; Serotec), GFAP (rabbit, Z0334; 1:200; Dako), IRF8 (goat, sc-6058; 1:200; Santa Cruz Biotechnology), GST-π (mouse, no. 610719; 1:200; BD Biosciences), NeuN (mouse, MAB377; 1:200; Chemicon), PMN (rat, MCA771GA; 1:200; Serotec), CD3 (145-2C11; 1:200; eBioscience), 5-HT (goat, no.20079; 1:200; ImmunoStar), platelet-derived growth factor receptor-β (PDGFRβ) (rat, no.136002; 1:200; BioLegend), and C5a (rabbit, BS-0324R; 1:200; Bioss). Infiltrating macrophages change their morphology to the amoeboid form in injured tissue (4, 60, 61). The range of morphologically amoeboid CD68 macrophages and 5HT+ area was measured using the BZ-II analyzer software program (Keyence). All images were captured using a BZ-9000 digital microscope system (Keyence), EVOS microscope (Life Technologies), or epifluorescence microscope equipped with a digital camera (BX51, Olympus).

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