Ninety minutes following the Late recall test, mice were transcardially perfused with 100-200 mL of 0.1M PBS (PBS) (pH = 7.4), followed by ∼100 mL 4% Paraformaldehyde (PFA) in PBS. The brains were removed and kept in 4% PFA until sectioning. Coronal brain slices of 50 μm thickness were obtained as described above.
Free-floating brain sections were rinsed (3 × 10 min) in PBS and incubated in blocking solution [PBS+5% normal goat serum (Biological Industries) and 0.3% Triton X-100 (Fluka analytical)] for 2 h at room temperature. Then, the sections were incubated with primary antibody against c-Fos [Anti-rabbit (Cell signaling) 1:500] at 4ºc in a shaking incubator overnight. The sections were then rinsed again in PBS (3 × 10 min) and treated with secondary antibody [Alexa fluor 594-conjugatedAnti-rabbit IgG antibody (Abcam), 1:500] for 2 h at 4ºc. After washing in PBS (3 × 10 min) at room temperature, sections were mounted on clean glass slides, air-dried, and cover slipped with DAPI + mountant (GBI labs).
Free-floating brain sections were rinsed (3 × 10 min) in PBS and incubated in blocking solution [PBS+5% normal goat serum (Biological Industries) and 0.3% Triton X-100 (Fluka analytical)] for 2 h at room temperature. Then, the sections were incubated with primary antibody against c-Fos [Anti-rabbit (Cell signaling) 1:500] at 4ºc in a shaking incubator overnight. The sections were then rinsed again in PBS (3 × 10 min) and treated with secondary antibody [Alexa fluor 594-conjugatedAnti-rabbit IgG antibody (Abcam), 1:500] for 2 h at 4ºc. After washing in PBS (3 × 10 min) at room temperature, sections were mounted on clean glass slides, air-dried, and cover slipped with DAPI + mountant (GBI labs).