CREB Immunofluorescence in Mouse Hippocampus

The test was conducted as described previously^{61 (link),62 (link)}. Brains were removed and then placed in 4% paraformaldehyde overnight. The fixed brains were washed with PBS and dehydrated in 5–30% sucrose at 4 °C for 48 h. Each brain (20 μm thick) was sectioned with a cryostat (MICROM HM 525, Walldorf, Germany), then sections were blocked in PBT (0.05% Tween 20 in 1X PBS) containing 0.3% Triton X-100 (Sigma) and 3% donkey serum (Genetex) for one hour at room temperature. The following primary antibodies were used for immunofluorescence: mouse anti-CREB polyclonal antibody (1:500, 35-0900, Invitrogen), and. Brain sections were incubated with the primary antibodies at 4 °C overnight, and washed three times in PBT. Brain sections incubated for 2 h with Alexa Fluor 647-conjugated donkey anti-mouse secondary antibody (1:500, Invitrogen). After the incubated brain sections were washed three times with PBT, the sections were covered with Vectashield HardSet Antifade mounting medium with DAPI (Vector Laboratories, H‐1500) and imaged with a confocal microscope (LSM800; Carl Zeiss). For cell counting, the measurement of the analyzing particles using the Image J software was used to count specifically immuno-stained cells in the hippocampus.