2.7. Chromatin immunoprecipitation (ChIP)

ChIP assays were performed according to the kit protocol (Santa Cruz Biotechnology, Visalia, CA, USA) with an anti-AhR antibody (Thermo Fisher Scientific, Cat. MA1–514, Rockland, IL, USA). The PCR product corresponding to the Prkn proximal gene promoter was generated from an aliquot of immunoprecipitated material. Brain homogenates from WT mice treated with a single oral dose of TCDD or vehicle (corn oil) were washed with PBS buffer and crosslinked with 1% formaldehyde. After chromatin isolation, the DNA was fragmented, and immunoprecipitation was performed. The crosslinking was reversed, the DNA was purified, and PCR amplification was performed as follows: initial denaturation at 94 °C for 3 min and 28 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, and extension at 72 °C for 30 s. A final extension cycle at 72 °C for 10 min was added to the end of the program. The oligonucleotides used for PCR amplification were 5′-AGAAGTGAGCAGGGGGTCGGG-3′ (forward) and 5′-AAGGACCTAC GCGGGCACTG-3′ (reverse).