cAMP ELISA
The level of cAMP in the hippocampus was measured by a direct competitive ELISA kit (Abcam, USA) as directed by the manufacturer. After weighing the frozen tissue, 0.1M HCl was added in a 5X volume. Then the sample was homogenized on ice and centrifuged at high speed for five minutes, collecting the supernatant. After loading the samples into 96-well plates, the samples were incubated for an hour with the cAMP antibody. The wells were incubated for an additional hour with cAMP- Horseradish Peroxidase (HRP) and washed three times with wash buffer before the final step. Thereafter, HRP developer was added and incubated for 1 hour. A 1 M HCl solution was used to stop the reaction, and the color developed was measured using an ELx800 plate reader (BioTek, USA).