single value for each animal. A mean6SE was calculated from the data in each group (n = 5–6 animals), and statistical analysis was performed as described below. Double Immunofluorescence Staining Coronal sections were incubated with 10% normal donkey serum for 1 h at room temperature in PBS containing 0.1% Triton X-100, followed by incubation with appropriate primary antibod- ies overnight at 4uC in the same buffer. The following primary antibodies were used in different combinations: rabbit anti-acetyl p53-Lysine373, mouse anti-NeuN (1:200, Millipore, Billerica, MA) and rabbit anti-Puma a/b (1:200, Santa Cruz Biotechnology, Santa Cruz, CA). After primary antibody incubation, sections were washed and incubated with Alexa Fluor594/647 donkey anti-rabbit or Alexa-Fluor488/594 donkey anti-mouse secondary antibodies (1:500; Invitrogen Corporation, Carlsbad, CA), respec- tively, for a period of 1 h at room temperature. Sections were washed with buffer, followed by PBS and then briefly, with water. Sections were mounted using water-based mounting medium with anti-fading agents (Biomeda, Fisher Scientific, Pittsburgh, PA). Negative experimental controls were incorporated by omission of the primary antibody, thus confirming the absence of non-specific immunofluorescent staining, cross-immunostaining, or fluores- cence bleed-through. Confocal Microscopy and Image Analysis Double-labeled images were captured on an LSM510 Meta confocal laser microscope (Carl Zeiss, Germany) using a 40X oil- immersion Neofluor objective with a 1.3 numerical aperture. The image size was set at 102461024 pixels, as previously published [40]. Excitation/emission laser filters settings were used for various chromophores as follows: argon/2 laser was used for Alexa Fluor488, with excitation maximum at 490 nm and emission in the range of 505–530 nm and HeNe1 laser was used for Alexa Fluor594, with excitation maximum at 543 nm and emission in the range of 568–615 nm. The captured images were viewed and analyzed using LSM510 Meta imaging software. Brain Homogenates and Subcellular Fractionations For brain tissue preparation, rats were sacrificed under isoflurane anesthesia after GCI, at the timepoints described in the figure legends. The CA1 region of the hippocampus was micro-dissected bilaterally from the hippocampal fissure and immediately frozen in liquid nitrogen. The protocol for obtaining total tissue lysates was as follows: tissue homogenization was performed with a Teflon-glass homogenizer in ice cold homog- enization medium consisting of 50 mM HEPES (pH 7.4), 150 mM NaCl, 12 mM b-glycerophosphate, 3 mM dithiotheitol (DTT), 2 mM sodium orthovanadate (Na3VO4), 1 mM EGTA, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% Triton X-100, and 10 mg/ml each of aprotinin, leupeptin, and pepstatin A. The homogenates were centrifuged at 15,0006g for 30 min. at 4uC and total lysate supernatants were collected and stored at 280uC for later use. Protein concentrations were determined by a Lowry protein assay kit with bovine serum albumin utilized as a standard. Co-Immunoprecipitation For co-immunoprecipitation (Co-IP), total fractions (each containing 200 mg of protein) obtained at the ischemic reperfusion timepoints described in the figure legends, were diluted 4-folds with 500 mL of HEPES buffer containing the following reagents: 50 mM HEPES (pH7.4), 150 mM NaCl, 10% glycerol, 1% Triton Acetylation of p53 X-100, and 1 mM each of EGTA, EDTA, PMSF and Na3VO4. The samples were then pre-incubated with 20 ml protein A/G and centrifuged to remove non-specific protein bound to the A/G protein. The supernatant was incubated with 5 mg of mouse anti- p53 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and mixed for 4 h at 4uC. After the addition of Protein A/G- sepharose, the mixture was incubated at 4uC for 2 additional hours. Samples were washed three times with HEPES buffer and eluted by sodium dodecyl sulfate-polyacrylamide gel electropho- resis (SDS-PAGE) loading buffer, then boiled for 5 min. Western Blots Western blotting was performed on total lysates collected post- reperfusion, as described in the relevant figure legends and in our laboratory publications [10]. The antibodies used were as follows: Rabbit anti-Acetyl p53 specifically recognizing p53 acetylation at Lysine373 by p300, (1:150, Millipore, Billerica, MA), Rabbit anti- Acetyl-p53 (Lys382, 1:200, Cell Signaling Technology, Inc.), Rabbit anti-Actin (1:300, Sigma-Aldrich, St. Louis, MO), rabbit anti-Mdm2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-p53 (1:200, Cell Signaling Technology, Inc., MA), rabbit anti-Puma a/b (1:150, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-Ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was then washed with T-TBS to remove unbound antibody, followed by incubation with Alexa Fluor 680 goat anti-rabbit/mouse IgG and Alexa Fluor 680 donkey anti-rabbit/goat IgG for 1–2 h at room temperature. Bound proteins were visualized using the Odyssey Imaging System (LI-COR Bioscience, Lincoln, NB) and semi-quantitative analysis of the bands was performed with Image J analysis software (Version 1.30 v; NIH, USA). Band densities for the indicated proteins were normalized and expressed relative to actin house- keeping gene total protein or total p53 protein, as indicated in the figures. Normalized means were then expressed as fold change of the corresponding value for control (sham operated) animals. A Mean6SE was calculated from the data, from 4–5 animals per treatment group for graphical presentation and statistical com- parison. Real Time-PCR Total RNA from the female rat hippocampal CA1 region was isolated following ischemic reperfusion as described in the corresponding figure legends and LightCycler RNA Amplification Kit SYBR Green I (Roche Applied Science, IN) was utilized. A negative control, which consisted of pooled total RNA run in the RT-PCR without RT added, was also included. Actin was used as a loading control. Five hundred nanograms of the RT reactions were used for the PCR, with the following primer sets to identify p53: forward primer 5’- TCT CCC CAG CAA AAG AAA AA - 3’ and reverse primer 5’ – CTT CGG GTA GCT GGA GTG AG – 3’. PCR reactions were conducted under the following conditions: 35 cycles at 95uC for 5 min, followed by 65uC for 30 min and 72uC for 3 min. at an annealing temperature of 65uC and 4 mM of MgCl2. A total of 5 mL of the PCR product was electropho- resed, visualized with ethidium bromide, and photographed for documentation. A standard curve was generated and results were quantified accordingly in rg/mL. Statistical Evaluation Four to five animals were used per treatment group. All values were expressed as the means6SE. Statistical analysis of the results was carried out by One-Way Analysis of Variance (ANOVA), followed by the Student-Newman–Keuls post-hoc test to determine group differences. When groups were compared to a control group PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e27039