[Zbrafish] Re: Bleaching embryos after In Situ Hybridization

[Burdine, Rebecca D]

Here is the blurb from our lab protocol which I think we cribbed from David Parichy's protocols?  

Part 4: Removing pigmentation in older embryos.
Embryos start developing pigmentation at around 1dpf. Embryos can be de-pigmented using hydrogen peroxide. Embryos can be bleached right after PFA fixation (before MeOH storage) or upon rehydration after MeOH storage. If you do it on the first day of the in situ, simply add 3 additional 5' PBT washes.

1.	Make a bleaching solution of 3% hydrogen peroxide and 1% KOH in dH20. You need 1ml of solution per tube of embryos to be bleached. Bleaching solution should be made fresh each time, since mixing the two reagents starts the chemical reaction!

100ul 30% Hydrogen peroxide (stored in large 4C) + 100ul 10% KOH (stored at RT) + 800ul Millipore water=1 mL

2.	Aspirate PFA (or PBT) and add 1 mL of bleaching solution to each tube, and leave the tubes open to allow for gas escape.  Typically 36-hpf embryos take 10-15 minutes, and 5-dpf embryos take ~45 min.  Check the tubes occasionally in case the bubbles from the peroxide reaction push any embryos out the top of the tube.  

3.	When the embryos appear sufficiently bleached (monitor this closely), aspirate the bleaching solution and rinse 2-3 times in PBT, then continue to MeOH storage or the in situ protocol. You may want to remove the solution with a plastic transfer pipet instead of with the vacuum because the embryos will be floating on top and may get accidentally sucked up easily. After a couple PBT washes, the embryos will eventually return to the bottom of the tube.


Note that the reaction times seem to depend upon the bleach, the temperature in the room etc.  Watch a test batch closely and the minute you can no longer see black specs in the epitube, stop the reaction.  Letting this go too long will chew up your embryos.  In our hands bleaching is better than PTU as it doesn't affect development and can be done after fixation.

Becky

---------------------------------------------------
Rebecca D. Burdine, Ph.D.
Assistant Professor
Dept. of Molecular Biology
Princeton University
Washington Road Mof 433
Princeton, NJ 08544 
 
Phone: (609) 258-7515
Fax: (609) 258-1343
Email: rburdine from princeton.edu
Admin Assistant: Cathy Falk (609) 258-1604
 

> -----Original Message-----
> From: zbrafish-bounces from oat.bio.indiana.edu 
> [mailto:zbrafish-bounces from oat.bio.indiana.edu] On Behalf Of 
> nonolow from gmail.com
> Sent: Wednesday, August 20, 2008 12:56 PM
> To: bionet-organisms-zebrafish from moderators.isc.org
> Subject: [Zbrafish] Re: Bleaching embryos after In Situ Hybridization
> 
> bleaching is a routine approach in frog embryos, you could 
> find some protocols and recipes in the book, Early 
> Development of Xenopus
> Laevis: A Laboratory Manual,
> be aware the time of bleaching, hydrogen peroxide could 
> breakdown the staining of in situ hybridization.
> good luck
> 
> 
> low
> 
> 
> 
> 
> 
> On Aug 19, 1:22 am, shellyleib... from gmail.com wrote:
> 
> 
> > Hello
> > I'm Shelly, a PhD student at Gothilf's lab from Tel Aviv 
> University. I 
> > wanted to ask if anyone knows how to bleach the pigment of 2dpf 
> > zebrafish embryos post whole mount In Situ Hybridization 
> (it was not 
> > raised in PTU water).
> > Many thanks in advance
> >
> > Shelly
> 
> 
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