Eclipse ni

Digital images of spinal cord sections were captured at 10× magnification using a Nikon Eclipse Ni fluorescent and brightfield microscope system (Nikon, Tokyo, Japan). Image analysis software (Image J; National Institutes of Health, Bethesda, MD) was used to quantify IBA1, GFAP and CCR2 immunostaining in a minimum of 4 randomly selected lumbar spinal cord sections [38 ] from each animal. For analysis of IBA1 and GFAP immunostaining, a square with a fixed area (200 × 200 mm²) was positioned in the medial, central, and lateral third of the dorsal horn. The number of pixels occupied by immunostaining within a defined threshold was measured. The upper and lower threshold optical densities were adjusted to match positive immunoreactivity and the determined thresholds were applied uniformly to all sections. Results from the three sampled regions of the dorsal spinal cord were summed for each section and expressed as total number of pixels (# pixels) in the area. For CCR2, sampling area was confined to superficial laminae (I–II) of the spinal cord [38 ]. The data were presented as fractional area reflecting the percent of the sampled area that fell within a defined immunodensity threshold. The individuals who quantified the images were blind to the experimental conditions.

Images of spinal cord sections were acquired at 10x magnification using a Nikon Eclipse Ni fluorescent and brightfield microscope (Nikon, Tokyo, Japan). The Image J software (National Institutes of Health, Bethesda, MD) was used to quantify TNFα immunostaining in the superficial laminae (I-II) of four randomly selected lumbar spinal cord sections from each animal. The data were presented as the fractional area, which reflects the percentage of the sampled area that fell within a defined density threshold. The individual who analyzed the images was blind to the experimental conditions.

The application of these two methods to cells was as previously described ^{14 (link),15 (link)}. Briefly, cells transferred onto glass slides were washed with PBST (PBS + 0.05% Tween 20) 3 times and permeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells are blocked with normal serum or 5% bovine serum albumin for 1 h, and then incubated with an appropriate dilution of primary antibodies at 4 °C overnight. After three washes with PBST for 15 min, cells are incubated for 1 h with horseradish peroxidase-conjugated secondary antibody at room temperature. Cells are then developed with chromogen (DAB) for 5–10 min at room temperature after three washes are counterstained with hematoxylin. The slides are dehydrated and mounted with liquid mounting media. For immunofluorescence detection, cells are processed with the same procedure as above but incubated for 1 h with fluorophore-conjugated secondary antibodies (usually diluted 1:400) instead and covered with mounting medium with 4’−6-diamidino-2-phenylindole (DAPI). All stained slides are examined and photographed using a Nikon fluorescence microscope (Nikon Eclipse Ni, Tokyo, Japan) with a Wide Zoom Camera.