Anti histone h3 tri methyl k9

Combination treatments were set up as described in the methods section. 1 × 10⁶ cells per IP were harvested and fixed in 1% formaldehyde. Cells were washed and chromatin was isolated; fixed samples were resuspended in LB1 buffer (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL, 0.25% Triton X-100) for 10 minutes, pelleted and resuspended in LB2 buffer (10 mM TrisHCl, pH8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 5 minutes, pelleted and resuspended in LB3 buffer (10 mM TrisHCl, pH8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% NA-deoxycholate, 0.5% N-laurylsarcosine). Cells were sonicated for appropriate time to obtain fragments of between 200-500 bp. Following sonication, 10% triton X-100 was added and chromatin was snap frozen for chromatin immunoprecipitation studies. 30 μL beads per IP were washed and blocked overnight at 4°C in sterile 0.5% BSA:PBS. Chromatin was immunoprecipitated with chosen antibodies (anti-histone H3 (acetyl K9), anti-histone H3 (mono methyl K9) and anti-histone H3 (tri methyl K9) (Abcam, UK)) and eluted using elution buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS). Chromatin was purified using the QIAquick PCR purification kit (Qiagen) as described in the manufacturer's instructions.

Immunohistochemistry was performed for the pre-synapse protein synaptophysin (SYP) and the post-synapse protein neurogranin (RC3) as an indirect measurement of synapse protein expression. For the measurement of the global epigenetic marks, we detected the immunoreactivity of the acetylated histone H2B in lysine 16 (H2BK16ac), trimethylated histone H3 in lysine 9 (H3K9me3), and the modified nitrogenous bases, 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC). The primary antibodies used were as follows: anti-synaptophysin (1:1000, Cat: SAB4502906, Sigma-Aldrich, St. Louis, MO, USA), anti-RC3 (1:250, Cat: NBP229349, Novus Biologicals, Littleton, CO, USA), anti-histone H2B acetyl K16 (1:2000, Cat: ab177427, Abcam, Cambridge, UK), anti-Histone H3 trimethyl K9 (1:2000, Cat: ab8898, Abcam, Cambridge, UK), anti-5-methylcytosine, (1:500, Cat: ab10805, Abcam, Cambridge, UK), and anti-5-hydroxymethylcytosine (1:700, Cat: ab106918, Abcam, Cambridge, UK). The secondary antibodies used were biotinylated goat anti-rabbit (SYP, 5-mC, H3K9me3), biotinylated donkey anti-goat (1:250), (RC3, H2BK16ac), and biotinylated goat anti-rat (5hmC) at a dilution of 1:250 (Jackson Immunoresearch, West Grove, PA, USA). The signal was amplified using the peroxidase method. Finally, the brain slices were mounted in a Neo-Mount medium (Cat: 109016. Merck, State of Mexico, Mexico).