Embryo Treatment Before Hybridization
Descriptions of all solutions can be found in Reagents and Recipes.
For all the solution changes in the following steps, the volume is ~1.2-1.4 ml and most (95%) of the previous solution has been removed. This range of volume is because I use a pasteur pipette to add the solutions (except the hybridization solution), filling the tube most of the way up but leaving some room for the liquid to slosh back and forth during rocking. If you can't stand this imprecision, do 1.00 ml changes.
I'm using these terms as defined:
Start with ~50 µl settled embryos in ethanol in a 1.5 ml eppendorf tube. By the time they enter the hybridization solution they will have doubled in volume from swelling in the aqueous buffers.
- 'Wash': add new solution to the tube, invert a couple of times, then let the embryos settle
- 'Rock': add new solution and put the tube on a nutator (rocking platform) the specified amount of time
- 'x' indicates the number of times to repeat
Now comes the proteinase K treatment. This is where you would substitute in the heat or acetone treatment if you want to preserve a protein antigen in order to be able to detect both protein and mRNA in your embryos. These alternative treatments are described in Simultaneous Detection of mRNA and Protein. My current stock of ProtK is at 10 mg/ml, and I use it at 1:1000 (10 µg/ml) final concentration.
- Rock 1x, ethanol, 5 minutes
- Rock 1x, 90% xylenes / 10% ethanol, 1 hour
- leave ~100 µl ethanol from the previous step in the tube, then just add the xylenes right on top
- rocking may go longer if lunch continues some extra time
- embryos become almost transparent and assume a yellow tint, presumably because the xylenes clears the tissue and makes the central yolk mass visible
- Wash 2x, ethanol; Rock 1x, ethanol, 5 minutes
- embryos become white again when the xylenes are removed
- Wash 2x, methanol; Rock 1x, methanol, 5 minutes
- Rock 1x, 50% methanol / 50% (PBT+ 5% formaldehyde), 5 minutes
- Wash 1x, (PBT+ 5% formaldehyde); Rock 1x, (PBT+ 5% formaldehyde), 25 minutes
- that's the first post-fixation: try to keep it to within 5 minutes of the target time
- Rock 4x, PBT, 5 minutes each
- PBT washes can continue longer, I'm just giving you the minimum here
- Rock 1x, (PBT+ proteinase K), 5-7 minutes
- Wash 2x, PBT; Rock 1x, PBT, 5 minutes
- the ProtK digestion is stopped just by washing it out, instead of using a glycine buffer
- Rock 1x, (PBT+ 5% formaldehyde), 25 minutes
- that's the second post-fixation
- Rock 4x, PBT, 5 minutes each
- Rock 1x, 50% PBT / 50% hybridization solution, 10 minutes
- Pre-hybridization: drain the previous solution and add 1 ml hybridization solution, then place the tube in a float in a 55° C. water bath. Pre-hybridize the embryos for at least 1 hour. During this hour, change the hybridization solution twice: once shortly after putting them in the water bath, then again after 30 minutes. Periodically invert the tube of embryos a few times to see if there is any clumping. At first, some temporary clumps may form, but they are easily broken up as you rock the tube by hand. The embryos should now have a translucent, pearly appearance, and take a longer time to sink and pile up on the bottom. Pre-hybridization can continue for 2-3 hours if you like.
- After this 1 hour pre-hyb, you can store the embryos at -20° C. for quite a while, days or weeks. This way you can quickly set up an in situ late in the day just like an antibody stain: simply reheat the embryos to hyb temperature, add the probe and go home. But as the trade-off for this convenience, storage in the hybridization solution seems to degrade the embryo morphology over time.
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