Embryo Fixation Protocol(2004 CTH modified from RG)

For cuticle preps:

1.  Scrape, pipette, brush, or otherwise add embryos to 1.5 mL tube with PBS.

2.  Remove supernatant and wash 1 or more times in PBS.

NOTE:  I use a syringe with the smallest available needle that will fit in the tubes.  I press the needle flush with the wall to minimize lost embryos.

3.  Make FRESH (dechorionates more consistently) 50 % bleach treat embryos for 3 minutes to dechorionate.  Embryos will start to float more and will stick more easily to the walls of the tube.

4.  Remove supernatant and wash 1 or more times in PBS.

5.  Add 500 mL PBS, add 500 mL methanol, and equilibrate for 2 minutes.  Embryos will gravitate more toward bottom now.

6.  Remove supernatant, add 500 mL methanol, add 500 mL heptane, and shake vigorously for 5 minutes to devitellinize the embryos.

7.  Remove both layers.

NOTE:  If using a plastic syringe, the heptane will begin to dissolve it slowly and make it progressively more difficult to use.  I typically designate one aqueous syringe, which graduates to a few uses as an organic syringe as it gets older and is then pitched once it is near worthless.

8.  Wash in methanol 1 or more times.

9.  Add FRESH (older stocks form precipitant that severely obfuscate embryos) 1:4 glycerol:acetic acid.  50-200 mL is enough for a “normal size” prep of a few hundred embryos.  Bake overnight at 60°C with cap on.

10.  Pipette onto slide.  Allow 1:4 glycerol:acetic acid to run off and evaporate and blot remainder off.  Add 2 drops Hoyer’s mount and cover.  If in a hurry, view and photograph with either darkfield or phase contrast microscopy.

11.  If time, place 4 small magnets on coverslip, and flatten overnight at 60°C.  Then, view and photograph with either darkfield or phase contrast microscopy.

For antibody staining:

1.  Scrape, pipette, brush, or otherwise add embryos to 1.5 mL tube with PBS.

2.  Remove supernatant and wash 1 or more times in PBS.  I use a syringe with the smallest available needle that will fit in the tubes.  I press the needle flush with the wall to minimize lost embryos.

3.  Make FRESH (dechorionates more consistently) 50 % bleach treat embryos for 3 minutes to dechorionate.  Embryos will start to float more and will stick more easily to the walls of the tube.

4.  Remove supernatant and wash 1 or more times in PBS.

5.  Make or find 10X PBX, 1 M EGTA, and water.  Mix in following proportions:  50:25:300, and store for current and future use.  Add 375 mL of this solution, 125 mL 37 % formaldehyde, and 500 mL heptane.  Spin end-over-end fairly fast for 20 minutes.  The formaldehyde fixes the embryos.

6.  Remove bottom aqueous layer, leaving 500 mL heptane (top) behind.

7.  Add 500 mL methanol and shake vigorously for 5 minutes to devitellinize the embryos.

8.  Remove both layers.

9.  Wash in methanol 1 or more times.

10.  Store in methanol at -20°C until ready for blocking and staining.  They will keep several weeks, but performance seems to gradually decline.

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