Immunofluorescence Analysis of Spinal Cord Pathology

Immunofluorescence analysis was accomplished as previously described [20 (link)–22 (link)]. Briefly, cryosections (10 μm thick; n = 3 per spinal cord; 5 spinal cords per group) from lumbar spinal cords in each group were incubated with rat anti-CD3 (1:500; BD Biosciences, NJ, USA), rat anti-myelin basic protein (MBP) (1:1000; Abcam), anti-ionized calcium binding adaptor molecule-1 (Iba-1) antibody (1:2000; Wako, Osaka, Japan), mouse anti-glial fibrillary acidic protein (GFAP; 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and/or rat anti-platelet endothelial cell adhesion molecule (PECAM)-1 (1:500; Santa cruz), mouse anti-albumin (1:500; Santa cruz), and rabbit anti-immunoglobulin G (IgG) (1:500; Abcam), mouse anti-occludin (1:500; Invitrogen, MA, USA), mouse anti-ZO-1 (1:500; Invitrogen) as primary antiserum and cyanine 3- and fluorescein-isothiocyanate (FITC)-conjugated mouse/rabbit/rat IgG antibody (1:200–1:500; Jackson ImmunoResearch, West Grove, PA, USA) as secondary antiserum. Images from each section were captured using confocal imaging system (LSM 5 PASCAL; Carl Zeiss, Oberkochen, Germany) and their intensity quantified. Immunohistochemical analysis for Iba-1 was accomplished and analyzed as previously described [20 (link)–22 (link)].