In situ Protocol for embryos and juveniles of Convolutriloba longifissura
Lab/Group: Martindale Lab (Kewalo Marine Laboratory)
Related Journal & Article Information
Journal: Nature
Article Title: Acoel development indicates the independent evolution of the bilaterian mouth and anus
There is also a NatureNews story about this work: Getting to the bottom of evolution.
Introduction
This protocol is based on a in situ hybridization protocol used for embryos of the sea anemone Nematostella vectensis (Martindale et al 2004).
Materials
Reagents
Hybe Buffer (40 mL)
10x PBS = 18.6 mM NaH2PO4 (2.56 g NaH2PO4-H2O per liter dH2O)
84.1 mM Na2HPO4 (11.94 g Na2HPO4-H2O per liter dH2O)
1,750 mM NaCl (102.2 g NaCl per liter dH2O)
PTw = 1x PBS + 0.1% Tween-20 detergent
PBT = 1x PBS + 0.2% Triton X-100 + 0.1% BSA (store at 4 degrees C)
20x SSC = 0.3 M Na citrate + 3 M NaCl, pH 7
Alkaline Phosphatase buffer (50mL)
AP Substrate Solution
To AP buffer, add 3.3 μl/ml NBT (stock: 50 mg/ml in 70% dimethyl formamide:30% water) and then 3.3 μl/ml BCIP (stock: 50 mg/ml in dimethyl formamide). Keep this solution dark.
Equipment
Rocker table at RT and at 4 degrees
Procedure
Fixation
Fixative: 3.7% formaldehyde, in Sea water (make up fresh)
1. Starve Adults for two days. Fix hatchlings on the same day they hatched.
2. Relax adults and juveniles in 7.14% MgCl2 up to 10 min
3. Fix animals in 3.7% formaldehyde only for 4 hours at 4°C.
5. Wash 5x in PTw, 1x in dH2O, and transfer to fresh 100% MeOH
6. Replace MeOH 2x, store at -20°C in a screw cap tube
In situ hybridization
Use RNAse-free equipment and solutions through hybridization step. All washes are 5 min. at RT on rocker table unless otherwise stated.
DAY 1
Pretreatment
1. Transfer embryos to a 24 well dish and use 500μl for each wash.
2. Rehydrate through: 60% MeOH/40% PTw, 30% MeOH/70%, PTw 4 x PTw washes
3. Digest with Proteinase-K (0.01 mg/ml in PTw – make fresh) for 2-3 minutes (no shaker). (Use 4 µl of a 20 mg/ml stock in 8 ml)
4. Stop digestion with 2 (PTw + 2 mg/ml glycine) washes.
5. Wash with 1% triethanolamine in PTw
6. Add 1.5 μl acetic anhydride to 500 µl 1% triethanolamine, Vortex it an put on the probe for 5 minutes.
7. Take the solution, add 1.5 μl more acetic anhydride, vortex again and put it again on the probe.
8. Wash briefly in Ptw, then wash 2 × 5 min in PTw
9. Refix in 3.7% formaldehyde in PTw for 1 hour at RT.
10. Wash 5 x in PTw
11. Heat animals for 10 min at 80°C to destroy endogenous phosphatases
Prehybridization
12. Remove as much liquid as possible without letting the embryos falling dry, and add 500 μl hybe buffer B - incubate for 10 minutes at RT.
13. Remove liquid - add 500 μl hybe buffer. Place at hybe temp overnight
14. Wash embryos once with prewarmed Hybe to get rid of traces of the dissolved egg cluster jelly
Hybridisation
15. Dilute probe to a final concentration of 3-0.05 ng/μl (usually 2.0 ng/μl) in hybe solution (dig-labeled probe should be stored as a 50 ng/μl stock in hybe buffer at -20 degrees). Denature probe at 80-90°C max for 10 minutes.
16. Remove prehybe and add probe to each well. Hybridize overnight or the weekend.
DAY 2
17. Remove Probe (can be reused 4-5 times)
18. Wash 1 x for 10 minutes and 1 x for 40 minutes with hybe buffer at hybe temp. (Do not forget to prewarm hybe buffer)
19. Wash usung the following steps:
- 30 min in 75% hybe + 25% 2X SSC at hybe temp
- 30 min in 50% hybe + 50% 2X SSC at hybe temp
- 30 min in 25% hybe + 75% 2X SSC at hybe temp
- 30 min in 100% 2X SSC at hybe temp
- 3 × 20 min in 0.2X SSC at hybe temp
- 10 min in 75% 0.2X SSC + 25% PTw at RT
- 10 min in 50% 0.2X SSC + 50% PTw at RT
- 10 min in 25% 0.2X SSC + 75% PTw at RT
20. 10 min in 100% PTw at RT
Visualization of Probe
21. Wash 3 x with PBT at RT
22. Block in Boehringer-Mannheim Blocking buffer (diluted to 1x with maleic acid buffer) 1 hr at RT on rocker.
23. Incubate with Boehringer-Mannheim anti-Dig/AP (diluted in blocking buffer to 1:5000) at 4°C overnight on rocker.
DAY3
24. Wash 10x (or more) for 20-30 minutes in PBT.
25. Wash 3 x for 10 minutes in AP buffer (embryos tend to stick a lot).
26. Develop in AP substrate solution (make fresh) at RT in dark. Monitor color development. (Can also develop slower at 4 degrees)
27. Stop reaction by washing 5 x with PTw.
Troubleshooting
Critical Steps
Anticipated Results
References
Martindale, M. Q., Pang, K., & Finnerty, J. R. Investigating the origins of triploblasty: 'mesodermal' gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa). Development 131, 2463-2474 (2004).
Acknowledgements
Many thanks go to Kevin Pang for optimizing the in situ protocol in Nematostella and for the help adjusting it to acoel flatworm embryos.
Keywords
fixation, in situ hybridization, acoel, embryos