Mouse experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee and the University of California, Irvine. 15 month old male 3xTg-AD mice [15
] or non-transgenic age, strain and sex-matched mice were exposed to a 12-minute bilateral common carotid artery occlusion as previously described [28
]. The mice were sacrificed and the brains collected 24 and 48-hours post injury (n=4 sham and 4 ischemic, each). 12-month old mice were sacrificed 3-months post-ischemia following perfusion with PBS (3xTg-AD n=12 sham and n=14 ischemic, Control n=4 sham 4 ischemic).
Half brains were cut sagittally, without the hindbrain, and homogenized in T-per extraction buffer as previously described [10
]. Protein concentrations were determined by the Bradford method. The second half of the brains were placed in 4% paraformaldehyde for 48-hours then cut into 50 μM sections using a vibratome.
Western blots were performed as previously described [10
]. Western blots were normalized to GAPDH and represented as a percentage of sham. All error bars represent the standard error of the mean. We directly analyzed 4 sham animals and 4–5 ischemic animals for western blots.
were measured as previously described using a sensitive sandwich ELISA system [10
Light-level immunohistochemistry was performed with an avitin-biotin immunoperoxidase technique (ABC kit; Vector Laboratories Inc, Burlingame, CA) and visualized with diaminobenzidine as previously described [10
]. Fluorojade staining was performed as previously described [14
Fluorescent immunolabeling was performed using free-floating sections as previously described [10
]. The prevent signal bleed-through, all fluorophores were excited and scanned separately using lambda strobing.
Primary antibodies were used at 1:1000 unless otherwise noted and are listed below. HIF1α(Abcam Cambridge, MA), HIF1β(Abcam), HIF2α(Abcam), Synaptophysin(Sigma-Aldrich, St. Louis MO), VEGF (Abcam), GAPDH(1:10,000, Santa Cruz Biotechnology, Inc. Santa Cruz, CA), HSP90(Abcam), HSP70(Calbiochem, San Diego, CA), HSP60(Abcam), Casp3(Promega Madison, WI), HT7(1:3000, Pierce Biotechnology, Rockford, IL), AT8 (Pierce Biotechnology), AT100(Pierce Biotechnology), AT180 (Pierce Biotechnology), AT270(Pierce Biotechnology), PHF1(Kind gift of P. Davies), Thy1 (Millipore, Billerica, MA), 6E10(Signet, Dedham, MA), CT20(1:5000, Calbiochem), ADAM10(Calbiochem), ADAM17(Calbiochem), BACE1(Calbiochem), PS1(Novus, Littleton, CO), PS2(Novus), Aph1A (Novus), pAPPT668(Cell Signaling Danvers, MA), A H3(Millipore, Billerica, MA), T H3(Millipore), A H4 L8(Cell Signaling), Fe65(Millipore), MAPK(Cell Signaling), pGSK3β(Cell Signaling), tGSK3αβ(Cell Signaling), pAKT(Cell Signaling), tAKT(Cell Signaling)
Data were analyzed by Student’s t-tests. Outliers greater than 3x the standard deviation of the mean were removed from statistical analysis. Error bars were standard error of the mean. Results were considered significant only when p<0.05.