Ultrahyb ultrasensitive hybridisation buffer

Digoxigenin (DIG)-labeled antisense and sense RNA probes for zebrafish PP1 and PP2 mRNAs were synthesized using the DIG RNA labeling kit (Roche, Basel, Switzerland). Sections were pretreated with proteinase K and hybridized with each RNA probe in ULTRAhyb Ultrasensitive Hybridisation Buffer (Ambion, Life Technologies, Carlsbad, California, USA). The probe on the sections was detected by incubation with alkaline phosphatase-conjugated anti-DIG antibody (Roche) followed by a blue 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium color reaction.

Northern-blot analysis were performed as previously described39 (link). Briefly, three micrograms of total RNA in denaturing buffer were loaded per lane and run on denaturing formaldehyde 1.5% agarose gel in MOPS buffer. RNA were transferred to nylon membrane (IMMOBILON-NY+, Millipore corporation) by capillary transfer in 10X SSC buffer (Saline Sodium Citrate 1X: 150 mM NaCl + 15 mM sodium citrate) and then cross-linked to the membrane by UV irradiation. Membrane were hybridised at 42 °C with 5′-biotinylated oligonucleotide probes (5 nM) for either comEA (comEA2-NB) or mreB (mreB-NB) in ULTRAhyb Ultrasensitive Hybridisation Buffer (Ambion, Austin, TX). Following overnight hybridisation the membranes were washed twice in 2X SSC buffer containing 0.1% SDS at 65 °C according to the ULTRAhyb manufacturer instructions. Probed membranes were revealed using horseradish peroxidase-conjugated streptavidin and enhanced luminol substrate (Chemiluminescent Nucleic Acid Detection Module, Pierce, Rockford, IL). Luminescence signals were acquired using an imaging workstation equipped with a charge-coupled device camera (Thermo).

Brains were isolated from animals. The samples were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB, pH 7.4) overnight at 4 °C. Each organ was cryoprotected by immersion in 0.1 M PB containing 15% and 30% sucrose, embedded in OCT compound (Sakura) and sectioned at 20 μm on a cryostat.
Digoxigenin-labelled antisense RNA probes for lamprey bPPL and P-opsin were synthesised using a DIG RNA Labelling Kit (Roche Applied Science), as previously reported^{13 (link),46 (link)}. In brief, sections were pre-treated with proteinase K and hybridised with the antisense RNA probe diluted in ULTRAhyb-Ultrasensitive Hybridisation Buffer (Ambion) at 68 °C overnight. The probe was detected using alkaline phosphatase-conjugated anti-digoxigenin (RRID: AB_2734716, Roche Applied Science), followed by a blue 5-bromo-4-chloro-3-indolyl phosphate nitro blue tetrazolium colour reaction.