4.1 Animals and experimental protocols
Three months old adult male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) weighing ~30 g were used. Mice were housed under standardized conditions with free access to a standard chow and water. Mice were divided into two groups with 4 animals in each group. Group 1 was vehicle control. Group 2 was treated with JNK inhibitor SP600125 (LC laboratories, Woburn, MA). Control animals in group-1 (n=4) were given 250 μl of vehicle (45% w/v of 2-hydroxypropyl-β-cyclodextrin; Sigma Aldrich, St Louis, MO) by i.p injection once a day for continuous 14 days. Treated animals in group-2 (n=4) were given 250 μl of SP600125 (16 mg/kg/day of SP600125 in vehicle) by i.p injection once a day for continuous 14 days. Mice were sacrificed on day 15. One hemi-brain from each mouse was frozen for immunofluorenct staining (IFS). The other hemi-brain was used for biochemical studies. For IFS brain tissues were snap frozen with OCT (optimal cutting temperature) compound (Tissue-Tek-Cat. No. 4583; ThermoFisher Scientific, Atlanta, GA) at -70OC. The frozen brain tissue was cut on sagittal plane for sections by cryostat (MICROM HM 525, Thermo scientific, Atlanta, GA). All animal experiments were in compliance with the protocols approved by the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center at Fort Worth, in accordance with guidelines of the NIH.
4.2 Immunoblot analysis
Cortex from mouse hemi-brain (n=3) was homogenized for 30 seconds using a mechanical homogenizer with homogenization buffer (10 mM Tris 7.2, 150 mM NaCl, 5 mM EDTA, 1% Triton X100, 1% sodium deoxycholate, 5 mM sodium orthovanadate, 50 mM NaF,) containing proteinase inhibitors. The homogenate was incubated for 2-3 hrs with shaking at 4OC, sonicated for 10 seconds, and centrifuged at 12,000Xg for 30 minutes. The supernatant (protein extract) was used for determination of protein concentration using Biorad reagent. 40 μg of Protein extract was mixed with equal volume 2X SDS-PAGE loading dye solution containing β-mercaptoethanol and heated for 10 minutes at 90 OC. Proteins were separated by 16% SDS-PAGE and transferred to PVDF membrane at 200 mA for 3 hrs. The membranes were blocked with 2% BSA in TBST (10 mM Tris 7.5, 150 mM NaCl and 0.05% Tween 20) for 2 hrs in room temperature followed by overnight incubation with primary antibodies at 4OC. Following antibodies were used: Anti-PS1 (Cat. No. MAB5232; Millipore, Billercia, MA), anti-phospho-SAPK/JNK (Cat. No. 9255; Cell signaling Tech, Boston, MA), anti-JNK (Cat. No. sc-474; Santa Cruz Biotech, Santa Cruz, CA), anti-activated Notch1 (Cat. No. ab8925; Abcam, Cambridge, MA), anti-Hes1 (Cat. No. ab71559: Abcam, Cambridge, MA), and anti-βActin (Cat. No A5441; Sigma Aldrich Inc, St Louis, MO) The blots were developed by ECL system (Pierce, IL).
4.3 Reverse transcription-polymerase chain reaction (RT-PCR)
Homogenates from mouse cortex (n=3) were centrifuged and cell pellets were used to prepare total RNA using trizol reagent ( Invitrogen, CA) according to manufacturer protocol. First strand cDNA was synthesized using oligodT primers and reverse transcriptase (Invitrogen, CA). 34 cycles of PCR were performed with the primers for mouse Hes1 and GAPDH. 250 ng of cDNA was used in PCR for Hes1 and 62.5 ng of the same cDNA was used in PCR for GAPDH. PCR was carried out at 940C for 30s, 600Cfor 30s, 720 C for 60s. PCR products were run on a 5% poly-acylamide gel, stained with ehidium bromide, and visualized under uv light. The sequences for mouse Hes1 and GAPDH primers are (1) Hes1 forward: 5′-GCCAGTGTCAACACGACACCGG-3′ and Hes 1 reverse : 5′-TCACCTCGTTCATGCACTCG-3′ (2) GAPDH forward: 5′-AACTTTGGCATTGTGGAAGG-3′ and GAPDH reverse : 5′-TGTGAGGGAGATGCTCAGTG-3′
4.4 Immunofluorescent (IF) analysis
For immunofluorescent staining (IFS), each 10μm-thick cryosection was fixed in cold acetone, blocked with 10% donkey serum in TBST, and stained with optimum dilution of primary antibodies, then optimum dilution of fluorochrome-conjugated secondary antibodies. Primary antibodies were anti-presenilin-1 (Cat. No. MAB5232; Millipore, Billercia, MA), phospho-SAPK/JNK (Cat. No. 9255; Cell Signaling Tech, Boston, MA), anti-p53 (Cat. No. sc-98; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53 (Cat. No. 9284L; Cell Signaling Tech, Boston, MA), activated Notch1 (Cat. No. ab8925; Abcam, Cambridge, MA), and Hes1 (Cat. No. ab71559: Abcam, Cambridge, MA). Fluorochrome-conjugated secondary antibodies were Cy3-conjugated donkey anti-mouse IgG (Cat. No. 715-166-151; Jackson Immuno Research, Mill Valley, CA), Cy3-conjugated donkey anti-rabbit IgG (Cat. No. 711-166-152; Jackson Immuno Research, Mill Valley, CA), and Alexa-Fluor-488-conjugated chicken anti-goat IgG (Cat. No. A21467; Invitrogen, Carlsbad, CA). Antibody-stained immunofluorescent samples were mounted by anti-fading aqueous mounting medium containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and covered by cover slips. The magnification indicated in each figure shows that of the objective lens in Nikon Eclipse Ti-U fluorescent microscope. The ratio of % positive staining areas versus % DAPI regions was analyzed by NIH software image-J.
4.5 TUNEL Assay
For TUNEL assay, each 10μm-thick cryosection was fixed in 4% paraformaldehyde, permeabilized with 0.1% TritonX-100 and pH 7.2. Terminal transferase reactions (containing TdT and fluorescein-dUTP) were then performed with the in situ Cell Death Detection Kit (Cat. No. 11684795910; Roche, San Francisco, CA) for the TUNEL assay. Labeled samples were mounted by anti-fading aqueous mounting medium containing DAPI and covered by cover slips. The magnification in the figures shows that of the objective lens in Nikon Eclipse Ti-U fluorescent microscope.
4.6 Statistical analysis for IFS and TUNEL assay
For IFS and TUNEL assay, the statistical significance between any two groups was analyzed by unpaired Student’s t-test. If the F-test comparison of variance was less than 0.05 (i.e. nonparametric distribution), the unpaired t-test with Welch’s correction was used. Differences were considered statistically significant at values of p < 0.05. All measures of variance are presented as SEMs.