The percentage of r PKB/Akt showing nerves was determined by counting the neuronal pages that showed distinctive labeling in the DRG areas. The control group received same amount of car injection at same time as above. Immunofluorescence staining was performed following a procedures described by Ji et al.. Quickly, after identified success times, get a handle on and nerve injured rats were terminally anesthetized and perfused through the ascending aorta with saline, followed by four or five paraformaldehyde in 0. 1 M phosphate buffer. After perfusion, the L5 DRG and L5 back were removed and post fixed within the same fixative for 3 h and then replaced with 30% sucrose immediately. The transverse spinal sections and DRG sections were cut in a and processed for immunostaining with immunofluorescence. PF 573228 Most of the areas were blocked with 3% donkey serum in 0. Three full minutes Triton X 100 for 1 h at room temperature and incubated more than 2 days at 4 C with primary antibody. The sections were then incubated for 1 h at room temperature with Cy3 conjugated secondary antibody. For double immunofluorescence staining, the DRG sections were incubated with an assortment of anti phospho Akt antibody and neuroflament 200, Isolectin B4, and GFAP more than 2 times at 4 C. Except IB4 treated DRG sections, which were only treated by Cy3 conjugated secondary antibody, most of the above sections were treated by a combination of FITC and Cy3 Retroperitoneal lymph node dissection conjugated secondary antibody for 1 h at roomtemperature. The stained sectionswere examinedwith an IX71 fluorescence microscope and images were captured using a CCD place camera. The quantification of the immunofluorescence staining in the DRG was conducted by count the number of phospho PKB/Aktimmunoreactive positive neurons per section. In each rat, every fourth section was picked from the group of consecutive DRG sections, and four sections were measured for each DRG. The average percentage of r PKB/Akt IR neurons relative order Imatinib towards the total number of neurons were obtained for each animal across the different tissue sections, and then the mean_SE across animals was established. For spinal cord, the quantification was performed by measuring the area of g PKB/ Akt IR good staining in spinal dorsal horn of every area utilizing a digital image analysis system. A density threshold was set above back ground level firstly to recognize definitely stained structure. As positive area the area occupied by these structures was measured. In each rat, every fourth part was selected from a group of consecutive back sections, and six sections were measured for each rat. An average percentage of area of p PKB/Akt IR relative to the total area of the spinal dorsal horn of the sections was obtained for every animal from all 6 sections, then the mean_SE price across animals was established.