Rabbit anti pcreb

After the CPP test, four rats per group were randomly assigned to immunohistochemistry test. Rats were deeply anesthetized using 3% sodium pentobarbital i.p (30 mg/kg) and perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) intracardially. The brains were then removed and postfixed in 4% PFA immediately. After dehydration and paraffin embedding, the hippocampus was cut into 5-μm sections using a Leica RM2135 microtome. Immunohistochemistry was performed as described in product manual of streptavidin-biotin complex with peroxidase (Rabbit IgG) Kit (Boster, China). Antibodies for rabbit anti-p-CREB (1:150 dilution; Abcam, UK), rabbit anti-Nurr1 (1:50 dilution; Santa Cruz, USA), and rabbit anti-BDNF (1:150 dilution; Abcam, UK) were used to determine the levels of p-CREB, Nurr1, and BDNF. Five slices per rat containing hippocampus were selected, and then, three horizons of each slice were randomly observed in the light microscope (×40). Positive expression of p-CREB, Nurr1, and BDNF was defined as appearance of brown particles within the cell. We used Image-pro plus software (IPP 6.0, Media Cybernetics Inc., Silver Spring, Maryland, USA) to measure positive cells’ integrated optical density, and the relative content for p-CREB, Nurr1, and BDNF was represented by the mean value of each group.