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 pasteur pipettes are used to add the solutions (except the hybridization solution), filling the tube most of the way up but leaving some room for the liquid to move back and forth during rocking. It is also acceptable to use 1.00 ml changes.

Term definitions:

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 nearly doubled in volume from swelling in the aqueous buffers.

  1. Rock 1x, ethanol, 5 minutes.
  2. 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, up to 2 hours.
    • The embryos become almost transparent and assume a yellow tint, as the xylenes clears the tissue and makes the central yolk mass visible.
  3. Wash 2x, ethanol; Rock 1x, ethanol, 5 minutes.
    • The embryos become white again when the xylenes are removed.
  4. Wash 2x, methanol; Rock 1x, methanol, 5 minutes.
  5. Rock 1x, 50% methanol / 50% (PBT+ 5% formaldehyde), 5 minutes.
  6. Wash 1x, (PBT+ 5% formaldehyde); Rock 1x, (PBT+ 5% formaldehyde), 25 minutes.
    • This is the first post-fixation: try to keep it to within 5 minutes of the target time.
  7. Rock 4x, PBT, 5 minutes each.
    • PBT washes can continue longer, 5 minutes is the minimum.
Next, begin the proteinase K treatment. This is the step for which one can substitute a heat or acetone treatment, in order to preserve a protein antigen and be able to detect both protein and mRNA species in the embryos. These alternative treatments are described in Simultaneous Detection of mRNA and Protein. We store proteinase K (ProtK) at 10 mg/ml, and use it at 1:1000 dilution in PBT (10 µg/ml final concentration).

  1. Rock 1x, (PBT+ ProtK), 5-7 minutes.
  2. Wash 2x, PBT; Rock 1x, PBT, 5 minutes.
    • The ProtK digestion is stopped just by washing it out, instead of using a glycine buffer.
  3. Rock 1x, (PBT+ 5% formaldehyde), 25 minutes.
    • This is the second post-fixation.
  4. Rock 4x, PBT, 5 minutes each.
  5. Rock 1x, 50% PBT / 50% hybridization solution, 10 minutes.
  6. 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 by rocking the tube by hand. The embryos should now have a translucent, pearly appearance, and take a longer time to sink and pile up at the bottom of the tube. Pre-hybridization can continue for 2-3 hours, as desired.
  7. After a 1 hour pre-hybridization, the embryos can be stored at -20° C. for days or weeks. This allows one to quickly set up an in situ late in the day just like an antibody stain: simply reheat the embryos to hybridization temperature, add the probe solution and start the overnight hybridization. However, storage in the hybridization solution seems to degrade embryo morphology over time.

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