Michael Eisen Lab:Protocols:Kosman Fix

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" / (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

 * Methanol aliquot - 100ml
 * Embryo wash (dilute 10X stock in wash bottle)
 * mix several hours prior to use
 * 8ml of Embryo fix solution
 * 3ml fixation buffer
 * (1.3X PBS, 67nM  EGTA pH 8.0)
 * 1ml 37% formaldehyde
 * 4ml heptane

Procedure

 * 1) Prepare 8mL of embryo fix solution in a scintillation vial for each collection.
 * 2) *Add the fixation buffer and formaldehyde, followed by heptane
 * 3) Collect and age embryos.
 * 4) Dechorionate in 100% bleach on plate for 2.5 min.
 * 5) *Max of 2.5 min, less for more gentle fix
 * 6) *Embryos can also be bleached in the sieve, using pipette to wash bleach over embryos.
 * 7) 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.
 * 8) Transfer embryos from sieve into fix with paintbrush and shake for > 20 minutes at 1,500 to 2,000rpm.
 * 9) *Shake at higher rpm for higher embryo density in vial
 * 10) 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.
 * 11) Use Pasteur pipette and aspirator to rinse five times in methanol.
 * 12) 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?

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