For all immmunohistochemistry, animals were deeply anesthetized, transcardially perfused with 4% paraformaldehyde in 0.1 M Tris buffer, and the brains removed for post-fixation and sectioning at 75 um in the sagittal plane using a vibratome. Sections were quenched using a methanol solution containing 3% hydrogen peroxide for 15 minutes, placed in a blocking solution (2% BSA, 0.2% milk, 0.1% Triton X-100 in PBS) for an hour, and incubated in primary antibody overnight. The next morning, sections were placed in secondary antibody and reacted with ABC reagents (standard Vectastain ABC Elite Kit, Vector Labs, Burlingame, CA) for an hour in each solution. Finally, the sections were incubated in VIP reagent (Vector VIP substrate kit for peroxidase, Vector Labs, Burlingame, CA) to visualize immunoreactivity. For immunofluorescence, sections were placed in blocking solution for an hour, placed in monoclonal/polyclonal primary antibodies overnight, and incubated for an hour in secondary antibodies for fluorescent tagging. Photocomposites were combined in Photoshop primarily using the Photomerge function.
Antibodies: Activated caspase-3, Cell Signaling Technology Inc., Danvers, MA [1:1000]. Calbindin, Sigma-Aldrich, St. Louis, MO [1:1000]. NeuN, Chemicon, Billerica, MA [1:100]. Glial fibrillary acidic protein, Sigma-Aldrich, St. Louis, MO [1:400]. Glucocorticoid receptor, Santa Cruz Biotechnology, Santa Cruz, CA [1:8000]. Ki-67, BD Biosciences, San Jose, CA [1:800]. p53, Novocastra Laboratories, Newcastle upon Tyne, UK [1:1000]. Goat anti-rabbit biotinylated secondary antibody, Vector labs, Burlingame, CA [1:200]. Goat anti-mouse biotinylated secondary antibody, Vector labs, Burlingame, CA [1:200]. Goat anti-rabbit alexa fluor 488 secondary antibody, Molecular Probes, Carlsbad, CA [1:750]. Goat anti-mouse alexa fluor 594 secondary antibody, Molecular Probes, Carlsbad, CA [1:750]. Vectashield Mounting Medium with DAPI, Vector Laboratories, Burlingame, CA.