Ds ri1

Free floating sections were pretreated with peroxidase block and incubated for 1 h in goat serum using the ImmunoCruz staining system. Sections were then incubated overnight at 4 °C with rabbit anti-TH (1:1000). The following day sections were treated with the biotinylated secondary, the HRP-streptavidin complex and visualized with diaminobenzidine. Cells were counted as described previously [30 (link),65 (link)] using stereotaxic coordinates defined by Franklin and Paxinos [63 ]. Stained sections were visualized by light microscopy (Nikon Eclipse TE300, Nikon USA, Melville, NY). Images were taken with the Nikon DS-Ri1 and processed with the NIS-Elements 3.2 (Nikon). The substantia nigra boundaries were established as previously described [30 (link)]. Microphotographs for TH neurons were taken at a 40× magnification. Neurons were counted under a light microscope at a magnification of ×400. A neuron was counted if nucleus was visible and one or more clearly defined processes tapered gradually from the cell body. This number was considered to be representative of the number of dopaminergic nigral cells in each animal. The counts were taken of three individual sections from each mouse.

Fifty thousand 24-hour sera deprived cells/well were plated on type IV collagen-coated microporous polyester membrane (InnoCyte cell invasion kit, Calbiochem, San Diego, CA) with the following treatments: (5 μM 4-HPR (2095sc log and SEL cells), 10 μM 4-HPR (JSCC2 log and SEL cells), 2) 1 μg/ml tocilizumab, 3) 10 μM reparixin, 4) fenretinide+ tocilizumab, 5) fenretinide+ reparixin, 6) tocilzumab + reparixin, 7) fenretinide+tocilizumab+ reparixin, 8) DMSO control. Preliminary studies confirmed all cell line viabilities remained ≥96% and cell numbers remained comparable during all treatments. JSCC3 conditioned medium was used as the chemoattractant [15 ]. After 16 hours of invasion (37°C, 5% CO₂), cells were formalin fixed and stained with 0.1% v/v crystal violet solution. Images were captured by Nikon DS-Ri1 using NIS Elements (Nikon, Melville, NY), followed by target pixelation analyses by image segmentation [ImagePro software (Media Cybernetics, Inc., Rockville, MD)].

Chartings and high magnification drawings were made using an Olympus BH-2 microscope (Olympus, Center Valley, PA) equipped with a drawing tube. Low magnification section drawings were made with a Wild M8 stereoscope (Leica Microsystems, Buffalo Grove, IL) with attached drawing tube. Photomicrographs were made with a Nikon Eclipse E600 microscope using a Nikon DS-Ri1 digital camera controlled by Nikon Elements software (Nikon Instruments, Inc., Melville, NY). Multiple Z-axis focal planes were combined digitally. The brightness and contrast of the images were adjusted to match the view seen by the eye through the use of Adobe Photoshop (Adobe, San Jose, CA).