As the probe template, a 520-bp PCR fragment of the human NYX (nucleotide positions 1887-2406, GenBank accession number AJ278865; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) was subcloned into an SmaI-linearized vector (pBluescript II SK+; Stratagene) by blunt-end ligation, and a 551-bp fragment of the murine Nyx (corresponding to nucleotide positions 557-1107 in the human sequence) was subcloned into a PstI-linearized vector (pBluescript II SK+; Stratagene). The plasmid pRO4 containing the rat rhodopsin cDNA was kindly provided by Armin Huber, Institut für Zoologie, Universität Karlsruhe, Germany.
One microgram of plasmid DNA was linearized by restriction enzyme digestion and purified by phenol-chloroform extraction. Digoxigenin-UTP–labeled sense and antisense riboprobes were generated by in vitro transcription of linearized plasmids with a kit (DIG RNA Labeling Kit; Roche Molecular Biochemicals, Mannheim, Germany).
Before the in situ hybridization, sections were treated with proteinase K buffer (0.1 M Tris-HCl [pH 8] and 0.05 M EDTA) for 5 minutes at 37°C and digested with 0.3 μg/mL proteinase K (Sigma, Diesenhofen, Germany) for 8 minutes at 37°C. Slides were then washed two times for 3 minutes each in diethyl pyrocarbonate (DEPC)-treated water, postfixed for 15 minutes in paraformaldehyde (PFA; 4% PFA in 0.2 M PB), washed again three times with DEPC-treated water, and air dried.
Sixty microliters of hybridization solution (50% deionized formamide [Sigma], 5× SSC, 5× Denhardt’s solution, 0.5 mg/mL tRNA [Fluka, Buchs, Switzerland]) and 0.2 ng/μL of the digoxigenin-labeled riboprobe were denatured for 5 minutes at 80°C and applied to the sections. The slides were then incubated in a humidified chamber for 16 hours at 64°C. Posthybridization washing steps were performed two times for 30 minutes each in 0.1× SSC at 64°C. After a 10-minute wash in Tris-buffered saline (TBS; 0.15 M NaCl and 0.1 M Tris-HCl [pH 7.5]) at room temperature (RT), the slides were incubated for 30 minutes with blocking solution (10% blocking reagent in 0.1 M maleic acid, 0.15 M NaCl [pH 7.5]; Roche Molecular Biochemicals). Drained slides were incubated with alkaline-phosphatase–conjugated anti-digoxigenin antibody (1:500, in 10% blocking solution, 0.15% Triton X-100 in TBS; Roche Molecular Biochemicals) for 45 minutes at 37°C. Sections were then briefly rinsed two times for 15 minutes each in TBS and preincubated for 10 minutes in substrate buffer (0.1 M Tris-HCl [pH 9.5], 1 mM MgCl2, 10% tetramisole-hydrochloride [Fluka]). Four microliters nitroblue tetrazolium salt (NBT; 30 mg/mL; Bio-Rad Laboratories, Munich, Germany) and 4 μL 5-bromo-4-chloro-3-indolyl phosphate (BCIP; 15 mg/mL; Bio-Rad Laboratories) were mixed with 1 mL of substrate buffer, and each section was incubated in 200 μL of this solution, in a humidified chamber in the dark for 24 to 72 hours at RT. Color reaction was stopped with stop buffer (0.1 M Tris-HCl [pH 7.5] and 0.01 M EDTA), and covered with sorbitol (Merck, Darmstadt, Germany).