The proportion of Day 5 and Day 9 article inoculation for therapy groups was used as an indicator of tumor growth. N JNK I was kindly supplied by Dr. C. Bonny from University of Lausanne, Switzerland. After appropriate success situations, the animals were deeply anesthetized with isoflurane and perfused through the ascending aorta with saline followed by four to five paraformaldehyde k48 ubiquitin with 1. Five hundred picric in 0. 1 M PBS. After the perfusion, the L4 L5 spinal cord segments, L4, L5 dorsal root ganglions and skin with tumor size were eliminated and postfixed in exactly the same fixative overnight. DRG sections, spinal-cord sections, and skin sections were cut in a cryostat, and processed for immunofluorescence staining. In brief, the areas were blocked with a day later goat serum, and incubated overnight at 4 C with the next principal antibodies, GFAP antibody, Iba 1 antibody, pJNK antibody, g c Jun antibody, NeuN antibody, prodynorphin antibody, PKC antibody, PGP 9. 5 antibody, or ATF 3 antibody. The parts were then incubated for 1 h at room temperature with Cy3 or FITC conjugated secondary antibodies. The stained sections were examined with a Nikon fluorescence Human musculoskeletal system microscope, and images were taken with a CCD Spot camera. The pc Jun immunostaining was quantified by percentage of p c Jun constructive neurons in the DRG and by the depth of p c Jun immunofluorescence in the dorsal horn from three animals per group. Spinal cord and tumor mass were collected on day 9 post inoculation, to judge the JNK activation in tumor mass and spinal cord. The cells were processed for Western blots. As described previously, animals were quickly killed, and the L4 L5 spinal segments were quickly removed and homogenized in a SDS sample buffer containing an assortment of protease and phosphatase inhibitors. Protein samples were separated on SDS PAGE gel and used in polyvinylidene difluoride blots. The blots were incubated over night at 4 and blocked with five hundred milk C with antibody against phosphorylated JNK or GAPDH. These blots were further incubated with HRP conjugated secondary antibody, produced in ECL solution, and exposed onto Hyperfilm. pifithrin alpha Mice were imaged at day 5 and 9 post inoculation by IVIS 100 Bioluminescence Imaging System. Mice were anesthetized with a mixture of 1 and air. Five hundred of isoflurane and placed in prone position on the imaging platform, with the hindpaws taped to the platform for greater exposure of the tumor. Luciferase substrate N Luciferin in PBS was injected intraperitoneally 5 minutes before imaging. Pictures were obtained every five minutes for forty minutes with the exposure time including 5 to 10 seconds for every 5 minutes. Bioluminescence signals were quantified using Living ImageR application by drawing regions of interest over the tumor region to have the photons per 2nd over the regions. The amount of remaining hindpaw was assessed utilizing the plethysmometer, to assess the development of melanoma in situ. To help expand check always the histology of tumor cells, hindpaw skin with tumor mass were cut in a cryostat and sections were stained with hematoxylin and eosin.