TUNEL analysis

Cell apoptosis was monitored by TUNEL detection kit (NeuroTACS II in Situ Apoptosis Detection Kit, Trevigen, R&D) according to manufacture protocol. Slides containing 10 μm thick slices of rat brain (as it is described in the “Immunohistochemical analysis” section above) were deparaffinized by warming in the oven at 60 °C for 5 min, rehydrated in xylene, 100, 95 and 75% solutions of ethanol and then in PBS. The tissues were permeabilized by incubation with NeuroPore solution™ for 27 min at RT. Then slides were immersed in quenching solution for 5 min at RT. After wash with TdT labelling buffer™, each tissue was incubated with 50 μl labelling reaction mixture for 1 h at 37 °C in humidified chamber. After wash with TdT stop solution™, slides were incubated with Streptavidin-HRP solution for 10 min at RT in humidified chamber, washed with PBS and immersed in DAB solution for 5 min at RT. Slides were counterstained with Blue Counterstain for 1 min and blued with ammonium water for 1 min. After wash in running tap water, tissues were dehydrated in graded alcohol and cleared in Xylene. A 50 μl drop of HistoChoice^® Mounting Media (AMRESCO Inc.) followed by a cover slip was placed on each brain tissue. The positive control slides containing water-or oxycodone-treated brain sections were pre-treated with TACS-Nuclease solution™ for 30 min at RT in humidified chamber followed by incubation with labelling reaction. Cells with brown staining in nucleus were considered as TUNEL-positive cells. Images were taken using light microscope BX43F (Olympus). Cells from three to five fields for each brain region has been investigated. The results are presented as a percent of TUNEL-positive cells in rat brain tissues exposed to water (W) oxycodone (O) and in control slides. Data presented as mean value of TUNEL-positive cells (± SEM). To determine statistical significance, data was analyzed by Student’s t-test, and data with p value lower than 0.05 was considered to be statistically different.