Dpx mounting media

Free-floating sections were rinsed in PBS with 0.1% IgG-free bovine serum albumin (Jackson ImmunoResearch, West Grove, PA, USA) wash buffer and then incubated in a polyclonal antibody to c-Fos primary (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-52) in wash buffer with 0.3% Triton-X100 (Sigma-Aldrich) at room temperature for 48 h at 4°C. After rinsing in wash buffer, sections were incubated for 60 min at room temperature in biotinylated anti-rabbit IgG secondary antibody (1:600; Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA, USA), rinsed in wash buffer, and then incubated in an avidin-biotin horseradish peroxidase complex (1:200, Vectastain Elite ABC kit) for 60 min at room temperature. The sections were then rinsed in wash buffer and reacted in a 0.1 M Tris buffer (pH = 7.6) with 0.56 mM 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich) solution, 0.003% hydrogen peroxide and 63 mM nickel ammonium sulfate (Sigma-Aldrich). After 5 min, sections were rinsed in Tris buffer to stop the chromagen reaction. Immunostained sections were mounted onto glass slides (Adhesion Superfrost Plus Microscope Slides, Brain Research Laboratories, Newton, MA, USA), cover slipped with DPX mounting media (Sigma-Aldrich), and imaged using bright field microscopy.

The isoflurane anaesthetic and nitrogen 4.0 were obtained from Baxter and Linde (both Bratislava, Slovak Republic), respectively. The indium-tin oxide (ITO) glass slides and α-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Bruker Daltonics (Bremen, Germany). Acetonitrile, methanol, ethanol and water (all LC-MS grade) were obtained from Merck Millipore (Prague, Czech Republic). Trifluoroacetic acid (TFA), chloroform, sodium chloride, 9-aminoacridine (9-AA), haematoxylin solution, Gill no. 2 and DPX mounting media were obtained from Sigma-Aldrich (Prague, Czech Republic). Eosin Y (0.5%) aqueous solution was purchased from VWR Chemicals (Stribrna Skalice, Czech Republic). Hydrochloric and nitric acid (65%, Analpure) were obtained from FlukaBioChemika or Analytika Ltd., respectively (both Prague, Czech Republic). Deionized water (18.2 MΩ/cm) was prepared using a Milli-Q system (Millipore, Molsheim, France). Omni Slides with hydrophobic surfaces were purchased from Prosolia, Inc. (Zionsville, USA).

To examine changes in the density of DOR-ir in the hippocampal CA1 region, sections were processed using previously described methods (Milner et al., 2011 ). Briefly, tissue sections were rinsed in 0.1 M Tris-buffered Sal (TS; pH = 7.6) and blocked in 0.5% BSA in TS for 30 min prior to incubation in rabbit anti-DOR antibody (1:5000) in 0.1% BSA and TS for 24 h at room temperature (25 °C) followed by an additional 24 h incubation at 4 °C. Sections then were incubated in a 1:400 dilution of donkey-anti-rabbit IgG (Jackson Immunoresearch Laboratories, Cat# 711-506-152, RRID:AB_2616595) for 30 min and rinsed in TS. Next, sections were incubated in avidin-biotin complex (ABC; Vectastain elite kit, Vector Laboratories, Burlingame, CA) at half the manufacturer's recommended dilution for 30 min, washed in TS, and reacted in 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) in 3% H₂O₂ in TS for 3.5 min. Tissue sections then were rinsed in PB and mounted from 0.05 M PB onto 1% gelatin-coated glass slides. The slides were dehydrated through an ascending series of ethanol concentrations and coverslipped from xylene using DPX mounting media (Sigma-Aldrich).

Cell death was examined by terminal deoxynucleotidyltransferase biotin dUTP nick end labeling (TUNEL) assay (Roche, Mannheim, Germany), which contain an anti-dUTP antibody labeled with peroxidase (POD), according to manufacturer’s instructions. Slides were counterstained with methyl green to visualize total nuclei and then mounted with DPX Mounting Media (Sigma Aldrich, St. Louis, MO, United States). Negative controls without enzyme were processed in order to avoid false positive results (data not shown). Images were obtained under a light microscope (Nikon Eclipse TE2000-E, United States).