2.3. Nuclear Protein Extraction and DNMT Activity

On day 5 post-hatch, chicks were decapitated (head and neck) and then perfused via the carotid artery with hypotonic buffer (10 mM HEPES, pH 7.9, with 1.5 mM MgCl₂ and 10 mM KCl). Brains were mounted with Tissue-Plus O.C.T compound (Fisher HealthCare, Houston, TX, USA), snap-frozen in liquid nitrogen, and then sectioned in the direction from rostral to caudal in a cryostat at −10 °C into 500 μm thick coronal sections within 30 min post-perfusion. The PVN and ARC were collected at 7.4 and 5.4 interaural, respectively, based on the Kuenzel and Masson chicken stereotaxic atlas [24]. Nuclei were collected using sterile disposable biopsy punch instruments (1 mm; Braintree Scientific Inc., Braintree, MA, USA) and were immediately transferred to sterile microcentrifuge tubes containing 25 μL pre-mixed pre-extraction buffer provided in the EpiQuik Nuclear Extraction Kit I (Epigentek, Farmingdale, NY, USA) and stored at –80 °C until further processing. To ensure anatomical accuracy, the remaining brain section was photographed and anatomy confirmed via digital overlays containing the respective nucleus boundaries according to the Kuenzel and Masson chicken stereotaxic atlas [24]. The nuclear extraction procedure was performed with the EpiQuik Nuclear Extraction Kit I (Epigentek), following the manufacturer’s protocol. Protein concentration was determined with a Bradford assay (Bio-Rad, Hercules, CA, USA). The EpiQuik DNMT Activity/Inhibition Assay Ultra Kit-Colorimetric (Epigentek) was used to determine DNMT activity, following the manufacturer’s protocol with 10 μg of each of the nuclear extracts. The absorbance (optical density; OD) was detected at 450 nm using a M200 Pro Multi-Mode plate reader (Tecan, Männedorf, Switzerland). Specific activity was calculated as

Six to nine samples from each treatment group were used for statistical analysis after exclusion of those that exceeded a two-fold standard deviation (SD) from the average.