Michael Eisen Lab:Protocols:Kosman Fix
This protocol fixes embryos in preparation for RNA or protein in situ stains. It is not appropriate for ChIP experiments.
- 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)
- 1.5mL Eppendorfs (one for each fix)
- Glass scintillation vials
- Orbital shaker capable of 1,500 to 2,000 rpm
- Vacuum line (optional)
- Methanol aliquot - 100ml
- Embryo wash (dilute 10X stock in wash bottle)
- mix several hours prior to use
- 8ml of Embryo fix solution
- Prepare 8mL of embryo fix solution in a scintillation vial for each collection.
- Add the fixation buffer and formaldehyde, followed by heptane
- Collect and age embryos.
- 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.
- 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.
- 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
- 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.
- Use Pasteur pipette and aspirator to rinse five times in methanol.
- Transfer embryos to 1.5mL Eppendorf. Rinse one time with methanol and refill Eppendorf with methanol. Store at -20C.
- Who has experience with this protocol?
or instead, discuss this protocol.