2.7. Chromatin immunoprecipitation (ChIP) assay

In a separate set of animals, the target brain regions (CeA and BNST) at P11 were dissected from MS/No MS (n = 6/group) rat brains collected at PND11 by a punch method using a 19 gauge guided syringe (C310GA/TW/SP). Tissue was not pooled. The dissected regions were cross-linked in 1% paraformaldehyde for 15 min at 25 °C. This cross-linking reaction was stopped by the addition of freshly prepared glycine to a final concentration of 0.125 mol/l followed by four washes in cold 0.1 M phosphate-buffered saline. The tissues were then homogenized in a cell lysis buffer (1× PBS, 0.4% NP-40, 10 μl/5 ml complete protease inhibitors cocktail, Abcam, USA) and kept for 15 min at 4°C. The homogenate was first centrifuged at 200 ×g for 2 min to pellet extracellular debris, followed by centrifugation at 5500 ×g for 5 min of the supernatant. The pellets were suspended in the nuclear lysis buffer (50 mM Tris–HCl pH 8.0, 10 mM EDTA, 1% sodium dodecyl sulfate, protease inhibitor cocktail), and using the Sonicator (Branson, USA), the extracted chromatin was sheared to 200–300 bp for proper binding with antibody. The amount of DNA was quantified and a 1/5 part of lysate was aliquoted before immunoprecipitation (input), so as to compare the relative binding to the specific antibody in different samples before and after immunoprecipitation. The chromatin was then subjected to immunoprecipitation using antibodies against anti-acH3K14 (Abcam, USA) overnight at 4 °C. As an experimental control for the specificity of antibody binding, non-immune rabbit IgG (Abcam, USA) and a no-antibody immunoprecipitation were performed for each chromatin sample. Protein-DNA-antibody complexes were precipitated with Dynabeads@protein A (Invitrogen, USA) for 2 h at 4 °C, followed by two washes each in low salt, high salt, LiCl and 1× TE buffers. The precipitated protein-DNA complexes were eluted from the antibody with 1% sodium dodecyl sulfate and 0.1 M NaHCO₃, and then reversed-cross-linking was done in 0.3 M NaCl at 65 °C overnight. Proteins were digested with proteinase K for 1 h at 45 °C. The DNA were extracted and purified (Qiagen, USA). Primers directed to the gene promoter OXTR (F 5′-CTTGGAAGCAGGAGGTGAAG-3′ and R 5′-TTTCCCCAAGAACAAGAACG-3′) were used for amplification. Following PCR, the ‘Input’ and immunoprecipitated DNA amplification reactions were run on 1% agarose gels. Immunoprecipitated DNA normalized to ‘Input’ was obtained using Image J (NIH Software).