Separate from the animals used in the multiplex bead array immunoassay, experimental, vehicle control and naïve animals were deeply anesthetized and perfused with cold isotonic saline followed by 4 % paraformaldehyde via cardiac puncture. Brains were moved to 4 % formaldehyde for 6–8 h, then allowed to sit in 30 % sucrose/PBS at 4 ° C for another 12–16 h. Tissues were then blocked, cut on a Leica CM3050 S cryostat (Thermo Shandon, Inc.; Pittsburgh, PA) at 40 μm and stored in cryobuffer (30 % each of glycerol, ethylene glycol and water, 10 % 2 × phosphate buffer) until use. Free float fluorescent IHC labeling was conducted as previously described [
43]. Experimental and vehicle control samples had an
n = 4 for each cytokine/cell type combination. The antibodies used were as follows: rabbit anti-IL-18 (1:250; sc6179, Santa Cruz Bio, Santa Cruz, CA), mouse anti-NeuN (1:1000; MAB377, Chemicon, Temecula, CA), mouse anti–glial fibrillary acidic protein (GFAP)(1:1000; MS-280-P, NeoMarkers, Fremont, CA), mouse anti-cd11b (1:1000; CBL1512, Chemicon), mouse anti-rat endothelial cell antigen (RECA)(1:1000; ab9774, Abcam), mouse anti-von Willebrand factor (vWf)(1:100; MAB3442, Chemicon), rabbit anti-smooth muscle alpha actin (1:250, Abcam), and mouse anti-NG2 chondroitin sulfate (1:500; MAB5384, Millipore). For anti-NeuN, anti-GFAP, anti-cd11b, anti-RECA, anti-von Willebrand factor, anti-smooth muscle alpha actin and anti-NG2 chondroitin sulfate, a species-appropriate Alexafluor™ 594 fluorescent-tagged secondary (1:1000, Molecular Probes; Eugene, OR) was used for visualization. For anti-IL-18, a biotin-labeled secondary (1:1000, Vector Laboratories; Burlingame, CA) followed by an Alexafluor™ 488 fluorescent-tagged tertiary antibody (1:1000, Molecular Probes) was used for signal amplification. Tissue sections labeled with only secondary and tertiary antibodies were used as controls. All sections used were located between interaural 6.20 mm, bregma −2.80 mm and interaural 5.20 mm, bregma −3.80 mm [
44]. For experiments requiring cell counting, investigators were blinded from the conditions. Sections were viewed and digitally captured with an Olympus BX51 microscope equipped with an Olympus DP-70 high-resolution color CCD digital camera. An Olympus BX61 equipped with a DSU spinning disk confocal system and DP-70 CCD camera was used to confirm same cell co-localization. Images of 40 μm tissues were acquired using a z step interval of 1 μm and analyzed using Slidebook™ software. Publication images were compiled using Adobe Photoshop CS4 digital image software. To more easily view the nuclei in 10x photomicrographs, the blue channel had the unsharpen mask filter applied with the following parameters: Amount = 500 %, Radius = 2.6 pixels, Threshold = 13 levels. For all photomicrographs, color levels were evened using the ‘levels’ tool. Input levels (0–255) were normalized in the RGB channel as follows: highlight input levels were set at the peak of the image histogram (10x and 40x), midtone levels were set at 0.8 (10x and 40x) and shadow levels were set at 75 (10x) or at the edge of the histogram closest to 255 or at 180, whichever was greater (40x).