Rabbit anti zif268

To investigate immediate early gene expression in response to odorant exposure, mice were presented with a tea ball containing 100 µl of pure +lim or dec for 1 h, 1 h before sacrifice. A rabbit anti-Zif268 (1:1000, Santa Cruz Biotechnology) and a rat anti-BrdU (1:100, Harlan Sera-Lab) were used. The appropriate secondary antibodies, coupled to Alexa Fluor 633 and 488 (Invitrogen) were used for revelation of the different markers. BrdU/Zif268 density was calculated by combining the mean percentage of BrdU/Zif268 positive cells per group to individual density of BrdU positive cells. All cell counts were conducted blind with regard to the experimental group.

Free-floating sections were prewashed three times for 10 min with 0.1 M Tris buffer saline (TBS; Sigma-Aldrich, Oakville, ON, Canada). Sections were then incubated in a primary antibody solution containing 1:500 rabbit anti-zif268 (Santa Cruz Biotechnology, Dallas, TX, USA), 1:500 goat anti-doublecortin (Santa Cruz Biotechnology, Dallas, TX, USA) 0.3% Triton-X (Sigma-Aldrich) and 3% normal donkey serum (NDS; MilliporeSigma, Burlington, MA, USA) in 0.1 M TBS for 24 h at 4 °C. Sections were washed three times for 10 min in TBS and a further incubation of sections commenced in a secondary antibody solution containing 1:500 donkey anti-rabbit ALEXA 594 (Invitrogen, Burlington, ON, Canada), 1:500 donkey anti-goat ALEXA 488 (Invitrogen, Burlington, ON, Canada), 3% NDS and 0.3% Triton-X in 0.1 M TBS for 24 h at 4 °C. Following three final rinses with TBS, the sections were mounted onto microscope slides and cover-slipped with PVA DABCO.

Mice were anesthetized and transcardially perfused with ice cold 4% paraformaldehyde 80 min after the start of the retrieval session. Home cage mice, which did not undergo a retrieval session, were perfused on the same day, staggered between perfusions of fear conditioned mice throughout the day. Brains were dissected and postfixed overnight in 4% paraformaldehyde, then sunk in 30% sucrose for 3 d. Brains were sliced into 20-μm coronal sections on a cryostat. Free-floating tissue sections were rinsed three times for 15 min in PBS with 0.25% Triton X-100 (PBS-T), then transferred to a blocking solution of PBS-T with 10% normal goat serum for 1 h at room temperature. Sections were incubated in a primary antibody solution of rabbit anti-Zif268 (Santa Cruz, polyclonal; 1:3000), or rabbit anti-SynapsinI (ThermoScientific; polyclonal; 1:1000) combined with mouse anti-PSD95 (Pierce Antibodies; monoclonal; 1:500). Primary antibodies were diluted in the blocking solution, incubated at 4°C for 72 h, and rinsed three times for 15 min in PBS-T. Secondary antibodies (Jackson ImmunoResearch; goat anti-rabbit 549, 1:1500, goat anti-mouse 647, 1:500) were diluted in the blocking solution and applied to the sections for 2 h at room temperature. Sections were mounted on slides and cover-slipped using DAPI mounting media to label cell nuclei and stored at 4°C.