Localization of Lamprey Photoreceptor Genes

Preparation of the RNA probes and in situ hybridization were carried out as previously described [8 (link)]. Digoxigenin (DIG)- and fluorescein-labeled antisense and sense RNA probes for lamprey parapinopsin and parietopsin mRNAs were synthesized using the DIG RNA-labeling kit and fluorescein RNA-labeling kit (Roche), respectively. Sections were pre-treated with proteinase K and hybridized with each RNA probe in ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). The pineal sections hybridized with DIG-labeled probes were incubated with horseradish peroxidase (HRP)-conjugated anti-DIG antibody (Roche) and subsequently treated with the TSA plus DNP (HRP) system (Perkin Elmer), followed by incubation with Alexa 488-conjugated anti-DNP antibody. The fluorescein-labeled probes were detected through incubation with alkaline phosphatase-conjugated anti-fluorescein antibody (Roche) followed by a color reaction using the HNPP Fluorescent Detection Set (Roche).