δfosb

As described above, 1 day after the challenge test (∼P 76), rats were sacrificed by live decapitation and their brain samples were used for Western blotting of ΔFosB (1:200, Cell Signaling Technology, Danvers, MA) following the protocol described in Experiment 1.

Sequential sets of tissues through the CeA were placed in 1% sodium borohydride in 0.1M PB for 30 minutes in order to remove any remaining aldehydes. Tissues were then incubated in 0.5% bovine serum albumin (BSA) and 0.25% triton X-100 in 0.1M Tris buffered saline (TBS; pH of 7.6) for 30 minutes. Tissues were then extensively rinsed in 0.1M TBS and incubated overnight in a cocktail of antibodies against c-Fos made in rabbit (1:9000, CalBiochem), ΔFosB made in rabbit (Cell Signaling), CRF made in guinea pig (1:8000, Peninsula Labs), or ENK made in mouse (1:1500, Fitzgerald), as appropriate. Proteins were visualized using FITC conjugated donkey anti rabbit (1:200), TRIT conjugated donkey anti guinea pig (1:200), and Cy5 conjugated donkey anti mouse (1:200, all secondary antibodies from Jackson ImmunoResearch, West Grove, PA). Control tissues were processed in parallel with the omission of primary antibodies. Tissues at matched rostro-caudal levels were photographed for quantification with an Olympus BX51 (Tokyo, Japan), and captured with Spot Advanced Software (Diagnostic Instruments Inc, Sterling Heights, MI). For ΔFosB quantification, n=6 MCtrl, n=4 FCtrl, and n=5 MEtoh and FEtoh. For c-Fos quantification the stress(−) group contained n=5 MCtrl, MEtoh, and FEtoh, and n=4 FCtrl animals. The stress(+) group contained n=6 of all conditions.