Adult Sycon specimens were collected from fjords located near Bergen, Norway (+60° 27' 33", +4° 56' 1") during the reproductive season from May to September (2008 to 2011). For in-situ hybridization, samples were immediately fixed in 100 mM MOPS, pH 7.5; 0.5 M sodium chloride; 2 mM MgSO4; 4% paraformaldehyde; 0.05% glutaraldehyde over night at 4°C, stepped into and extensively washed in 70% EtOH and stored at −20°C until processing. Macro sections of sponges in 24 well plates (Nunc) were rehydrated and washed in PBS/0.1% Tween (PTw). Samples were pretreated with 7.5 μg/mL proteinase K for 10 minutes at 37°C, followed by quenching with glycine (2 mg/mL PTw). Acetylation was performed by serial treatment with 0.1 M triethanolamine containing 0, 1.5, and 3 μl/mL acetic anhydride. Re-fixation was done in 4% paraformaldehyde/0.05% glutaraldehyde in PBS for 1 h at room temperature, followed by extensive washing in PTw. Tissue was prehybridized as previously described
[33 (link)] in 2 mL-tubes for 90 to 180 min at 51°C. Probe hybridization was done with denatured RNA probe (0.1-0.3 ng/μL, approximately 1 kb) for 12 to 18 h at 51°C. Stringent washes were carried out at 55°C as following: 1 × 10 min in hybridization buffer; 2 × 10 min 50% formamide/4 × SSC/0.1%; 2 × 10 min 50% formamide/2 × SSC/0.1% Tween; 2 × 10 min 25% formamide/2 × SSC/0.1% Tween, followed by 3 × 15 min 2 × SSC/0.1% Tween at room temperature. Samples were transferred to maleic acid buffer and incubated in 2% (w/v) Blocking Reagent (Roche) for 60 min at room temperature. After overnight incubation with AP-coupled anti-Digoxigenin-Fab fragments (Sigma, 1:5,000) at 4°C, samples were washed in maleic acid buffer at least 6 × 30 min. Probe was detected using NBT/BCIP as substrate (Roche) with tissue equilibrated in alkaline phosphatase buffer (100 mM sodium chloride, 50 mM MgCl2, 100 mM Tris pH 9.5, 0.1% Tween, 1 mM Levamisole). The staining reaction (0.5 to 3 days) was stopped with PBS/0.5% Tween, samples were transferred to 100% glycerol for microscopy or ethanol-dehydrated and embedded in epoxy resin (Sigma) for sectioning. Pictures of whole mount samples and sections were taken using a Nikon DS-U3 microscope and processed in Photoshop.
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