Anti activated caspase 3

Five sections of the brain cortex and anterior horns of the spinal cord from each rat were randomly selected and subjected to immunofluorescence staining. The tissue sections were incubated overnight at 4 °C with anti-NF-200 (1:500; Abcam, Cambridge, MA, USA), anti-GAP-43 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), anti-BDNF (1:500; Abcam), anti-NF-κB p65 (1:500; Abcam), Anti-p75NTR (1:500; Abcam), anti-MBP (1:500; Abcam), anti-glial fibrillary acidic protein (GFAP, 1:200, Thermo Fisher Scientific, Waltham, MA, USA), anti-COX-2 (1:1000; BioVision, Milpitas, CA, USA), and anti-CD68 (1:100; Santa Cruz Biotechnology) antibodies, individually. After washing in PBS, the sections were incubated with TRITC/FITC-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. The sections were subsequently mounted on glass slides and coverslipped with Antifade Gel Mount Aqueous Mounting Media (Southern Biotech, Birmingham, AL, USA). Stained sections were viewed under a microscope at 200x magnification, and images were taken of three fields per tissue section. Areas stained positively for GFAP, MBP, and CD68 were analyzed using NIH Image software. The numbers of cells stained positively for GAP43, caspase-3, NF-200, BDNF, NF-kB p65, COX-2, Caspase-3, and p75NTR were counted.

Immunohistochemical staining with anti-CD4 (1:500, Abcam, Cambridge, MA, USA), anti-COX-2 (COX-2; 1:1000, BioVision, Milpitas, CA, USA), anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), anti-NF-κB p65 (1:500, Abcam), and anti-MBP (1:500, Abcam), and immunofluorescence staining with polyclonal anti-BCL-2 (1:200; Santa Cruz, Dallas, TX, USA), anti-TNF-α (1:1000; ProSci Inc., San Diego, CA, USA), anti-activated BAX (1:200; Santa Cruz), anti-GAP43, and anti-CD68/ED1 (1:100; Santa Cruz) were performed as previously described (Cui et al., 2004 (link)). Double-immunostaining was performed using anti-CD4 (1:500, Abcam, Cambridge, MA, USA, green) and anti-CCR3 (1:500, Abcam, Cambridge, MA, USA) to identify Th2-polarized cells. Primary antibody omission controls were performed as negative controls.
Five sections from the motor cortex and bilateral anterior horns of the spinal cord for each animal were randomly selected and images were photographed under 20× magnification in three vision fields per section. The numbers of cells positive for COX-2-, NF-κB p65, GAP43, BAX, BCL-2 and caspase-3 were counted (cells positive for GAP43, BAX, and BCL-2 were counted via immunofluorescence staining and cells positive for COX-2-, NF-κB p65, and caspase-3 were counted using a counter-staining method).