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The slices were then dewaxed and dehydrated, and microwave antigen retrieval (10 min) was undertaken. The slices were incubated with 3% H202 for 10 min. Blocking buffer was added and the samples were then incubated overnight at 4��C with primary antibody (rabbit anti-rat NGF, 1:150). The next day, the slices were incubated for 30 min at room temperature with a biotin-labeled secondary antibody followed by streptavidin http://www.selleckchem.com/products/SRT1720.html biotin�Cperoxidase complex working solution. The tissues were dehydrated, mounted and stained with hematoxylin. After color development with DAB, the slices were examined and photographed using light microscopy. Phosphate buffer solution (PBS, pH 7.4) replaced the primary antibody in control samples. The antibodies, immunohistochemistry kit and DAB were purchased from Boster Bio-engineering, Wuhan, China. Image analysis was performed with Image-Pro Plus5.0 software. The light source was kept stable and settings were kept constant during imaging. Four sections with typical morphology were selected and three randomly selected fields of view were examined on each section. The average absorbance (LA) of NGF-positive cells was calculated in each field using NISDR image processing software (Nikon, Japan). Fresh tissue (100 mg) was obtained from each of the four brain areas (cerebrum, midbrain, cerebellum http://en.wikipedia.org/wiki/MERTK and spinal cord) at each of the three development stages. The tissue was ground on ice, and blended with 1,000 ?L tissue lysate (containing 10 g/L sodium deoxycholate, 10 g/L SDS, 150 mM NaCl, 50 mM Tris, 1 g/mL leupeptin, 1 ?g/mL aprotinin, 1 ?g/mL pepstatin, 0.1 ?g/mL phenylmethanesulfonyl fluoride). The suspension was centrifuged at 12,000 rpm for 40 min at 4��C. Samples (10 g) of total protein collected from the supernatant were separated with 15% SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% BSA for 2 h. This was followed by overnight incubation with the primary antibody (NGF 1:400, ��-actin 1:2,000, Boster) at 4��C. The samples were then incubated with the secondary antibody (1:5,000) at 37��C for 2 h. The PVDF membrane and developer (0.1 mL/cm2) were added to the samples at room temperature for 1 minute, and sealed with preservation film. The membrane was then pasted on an X-ray film in a dark room, exposed and http://www.selleckchem.com/products/Adrucil(Fluorouracil).html developed. The exposure time was adjusted to obtain high definition bands. The relative gray levels of each band were measured using UTHSCSA Image Tool version 3.0software, and the relative expression levels were calculated using ��-actin as an internal control. Statistical analysis was performed using SPSS version 13.0 software (SPSS, Chicago, IL). Means and standard deviations (��SD) of measurements of distribution density were recorded. Single factor ANOVA was used to analyze differences between pairs of data. P