This protocol is based on a in situ hybridization protocol used for embryos of the sea anemone Nematostella vectensis (Martindale et al. 2004).
Method Article
In situ protocol for embryos and juveniles of Convolutriloba longifissura
https://doi.org/10.1038/nprot.2008.201
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This protocol is based on a in situ hybridization protocol used for embryos of the sea anemone Nematostella vectensis (Martindale et al. 2004).
Hybe Buffer (40 mL)
See figure in Figures section.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)
See figure in Figures section.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.
Rocker table at RT and at 4 degrees
Fixation
Fixative: 3.7% formaldehyde, in Sea water (make up fresh)
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
Transfer embryos to a 24 well dish and use 500μl for each wash.
Rehydrate through: 60% MeOH/40% PTw, 30% MeOH/70%, PTw 4 x PTw washes
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)
Stop digestion with 2 (PTw + 2 mg/ml glycine) washes.
Wash with 1% triethanolamine in PTw
Add 1.5 μl acetic anhydride to 500 µl 1% triethanolamine, Vortex it an put on the probe for 5 minutes.
Take the solution, add 1.5 μl more acetic anhydride, vortex again and put it again on the probe.
Wash briefly in Ptw, then wash 2 x 5 min in PTw
Refix in 3.7% formaldehyde in PTw for 1 hour at RT.
Wash 5 x in PTw
Heat animals for 10 min at 80°C to destroy endogenous phosphatases
Prehybridization
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.
Remove liquid - add 500 μl hybe buffer. Place at hybe temp overnight
Wash embryos once with prewarmed Hybe to get rid of traces of the dissolved egg cluster jelly
Hybridisation
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.
Remove prehybe and add probe to each well. Hybridize overnight or the weekend.
DAY 2
Remove Probe (can be reused 4-5 times)
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)
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 x 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
Visualization of Probe
Wash 3 x with PBT at RT
Block in Boehringer-Mannheim Blocking buffer (diluted to 1x with maleic acid buffer) 1 hr at RT on rocker.
Incubate with Boehringer-Mannheim anti-Dig/AP (diluted in blocking buffer to 1:5000) at 4°C overnight on rocker.
DAY3
4 - 5 days
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).
Many thanks go to Kevin Pang for optimizing the in situ protocol in Nematostella and for the help adjusting it to acoel flatworm embryos.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version