[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.
In-Situ Hybridization of Adult Sycon Sponge
[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.
Corresponding Organization :
Other organizations : University of Bergen
Protocol cited in 9 other protocols
Variable analysis
- Proteinase K treatment (7.5 μg/mL for 10 minutes at 37°C)
- Acetylation treatment (0.1 M triethanolamine with 0, 1.5, and 3 μl/mL acetic anhydride)
- Probe concentration (0.1-0.3 ng/μL, approximately 1 kb)
- In-situ hybridization signal detection
- Fixation conditions (100 mM MOPS, pH 7.5; 0.5 M sodium chloride; 2 mM MgSO4; 4% paraformaldehyde; 0.05% glutaraldehyde)
- Washing and rehydration steps (70% EtOH, PBS/0.1% Tween)
- Hybridization temperature (51°C)
- Stringent wash conditions (varying formamide/SSC/Tween concentrations and temperatures)
- Blocking and antibody incubation conditions (maleic acid buffer, 2% Blocking Reagent, anti-Digoxigenin-Fab fragments)
- Probe detection method (NBT/BCIP substrate)
- Not explicitly mentioned
- Not explicitly mentioned
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