Michael Eisen Lab:Protocols:Kosman Fix

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Overview

This protocol fixes embryos in preparation for RNA or protein in situ stains. It is not appropriate for ChIP experiments.

Materials

  • Embryo wash sieve
  • Pasteur pipettes <ref name="Wheaton number 357331" / </ref> (or vacuum line for aspirating waste)
  • Glass beaker for pipette waste (or vacuum line for aspirating waste)
  • Glass beaker for fix waste (or vacuum line for aspirating waste)
  • Paintbrush
  • 1.5mL Eppendorfs (one for each fix)
  • Glass scintillation vials

Equipment

  • Orbital shaker capable of 1,500 to 2,000 rpm
  • Vacuum line (optional)

Reagents

Procedure

  1. Prepare 8mL of embryo fix solution in a scintillation vial for each collection.
    • Add the fixation buffer and formaldehyde, followed by heptane
  2. Collect and age embryos.
  3. Dechorionate in 100% bleach on plate for 2.5 min.
    • Max of 2.5 min, less for more gentle fix
    • Embryos can also be bleached in the sieve, using pipette to wash bleach over embryos.
  4. Rinse embryos into sieve (if bleached in the plate) with embryo wash. Then wash with diH2O for several minutes. Collect embryos in center of sieve basket.
  5. Transfer embryos from sieve into fix with paintbrush and shake for > 20 minutes at 1,500 to 2,000rpm.
    • Shake at higher rpm for higher embryo density in vial
  6. Remove bottom phase and add 8mL methanol. Shake vigorously by hand for one minute. Aspirate off the top phase, then the bottom, leaving the devitelliniezed embryos in the vial.
  7. Use Pasteur pipette and aspirator to rinse five times in methanol.
  8. Transfer embryos to 1.5mL Eppendorf. Rinse one time with methanol and refill Eppendorf with methanol. Store at -20C.


Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


References

Bibliography

Notes