Immunohistochemistry (IHC)
Fluorescent IHC for CD4, CD154, and GFAP was performed on the L5 segment of lumbar spinal cord sections following a previously published procedure [13]. For CD4, a FITC-rat-anti-mouse CD4 mAb (clone RM4–5; 1:100) (BD Biosciences, San Diego, CA), was used; for CD154, a primary mAb, purified Armenian hamster-anti-CD154 (clone MR1; 1:100) (BD Biosciences), and a secondary antibody (Ab), TRITC-goat-anti-Armenian hamster IgG (1:5000) (Jackson Immuno Research Laboratories, West Grove, PA), were used. For GFAP, a primary Ab, polyclonal rabbit-anti-GFAP (1:10,000) (DAKO UK Ltd., UK) and a secondary Ab, Cy3-goat-anti-rabbit IgG (1:800) (Jackson Immuno Research Laboratories), were used. DAPI-containing mounting media (either VECTASHIELD® (Vector laboratories, Burlingame, CA) or Fluoromount-GT (VWR, Bristol, CT)) was used. “Non-stained” and “no primary Abs” controls were included when performing IHC, and no significant fluorescent signal was detected from these controls. For CD4 and CD 154 IHC, images were taken with an Olympus fluorescence microscope (U-ULH) (Olympus Optical Co.) and an Olympus Q-FIRE camera. For GFAP IHC, slices were examined with a Nikon Eclipse E800 fluorescence microscope (Nikon Instruments Inc., Melville, NY) and a SPOT RT Slider CCD microscope digital camera (Burlingame, CA). To determine GFAP expression in the spinal cord dorsal horn, black and white images were analyzed using Image J (NIH). Both the total dorsal horn area and GFAP immunostaining intensity were measured for each L5 dorsal horn image (represented images shown in Figure 5A). A ratio of total intensity/total dorsal horn area was then calculated and used as the GFAP immunoreactivity for each L5 dorsal horn slice. The average GFAP immunoreactivity of three slices from the same tissue sample were calculated and used as the GFAP expression for that particular sample in the final data analysis. The experimenter performing the analyses was blinded to the experimental groups.