Dechorionation of zebrafish embryos
prajakta dongre
I want to dechorionate zebrafish embryos by pronase
treatment. I have some questions regarding the same.
Following are the questions.
1) Is pronase and proteinase K same?
2) From where pronase can be order, Is it Sigma or
Merck or which company?
3) How is pronase stock prepared, is it dissolved in
water or which buffer of what pH?
4) What is the working concentration of pronase for
dechorionation?
5) Any other suggestions regarding dechorionation is
welcome.
Thanking you
Yours sincerely
Prajakta Limaye
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Burdine, Rebecca D
Hi Prajakta,
> 1) Is pronase and proteinase K same?
No no no no no!
For pronasing be sure to use Pronase (Protease, Type XIV: Bacterial,
from Streptomyces griseus) Sigma P5147-1G
> 2) From where pronase can be order, Is it Sigma or Merck or
> which company?
See above.
> 3) How is pronase stock prepared, is it dissolved in water or
> which buffer of what pH?
We prepare 10mg/ml stocks in dH2O. We store 2mls in a 15 ml conical
tube and freeze at -20 until needed for dechorionation.
This is from our injection protocol - although we never dechroinate to
inject anymore.
5. To pronase embryos collect a 500ml beaker filled with blue water, a
tube of pronase stock, your glass pipette, a 60mm dish and a pair of
forceps.
6. Dilute pronase stock 5x with egg water. Transfer the embryos to the
bottom of a 60mm dish and remove as much of the egg water as possible.
Add the diluted pronase to the dish and swirl the embryos.
7. Monitor the embryos carefully. Use forceps to pinch the chorions.
If a pinch collapses the chorion the pronase has worked long enough.
Quickly pour them into a 500ml beaker with egg water. Swirl the beaker,
let embryos settled to the bottom and pour off the blue water.
9. Rinse the embryos into another 300 mls of blue water. Swirl and
let the embryos sink to the bottom. Decant as much egg water as possible
without disturbing the embryos. DO NOT LET EMBRYOS CONTACT AIR or they
will explode.
10. Repeat the rinses in egg water for a total of 3-4 rinses. The
chorions should come off of the embryos during the swirling. Once the
embryos are out of their chorions, use the glass pipette to remove them
to a glass dish containing embryo media.
The embryos can only touch GLASS or AGAR COVERED PLASTIC after the
chorions are removed.
Note that too much pronase will cause all the embryos to turn to mush as
you do the rinsing. We dispense blue water from a 20L carboy so the
force of the flow helps blow off the chorions somewhat.
This protocl is meant for embryos at the 1-2 cell stage. Some people
use this for later stages, but once the embryos are done with epiboly, I
find it easier to use forceps than deal with the finicky pronase.
Each batch of pronase will act differently. Sometimes it takes seconds
for chrions to become flimsy, other times it can take a minute. You
have to watch the embryos closely once they are in pronase.
Good luck,
Becky