Bleaching embryos after In Situ Hybridization

[shelly...@gmail.com]

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

[non...@gmail.com]

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

[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: rbur...@princeton.edu
Admin Assistant: Cathy Falk (609) 258-1604

> -----Original Message-----
> From: zbrafish...@oat.bio.indiana.edu
> [mailto:zbrafish...@oat.bio.indiana.edu] On Behalf Of
> non...@gmail.com
> Sent: Wednesday, August 20, 2008 12:56 PM
> To: bionet-organi...@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...@gmail.com wrote:
>
>

> _______________________________________________
> Zbrafish mailing list
> Zbra...@net.bio.net
> http://www.bio.net/biomail/listinfo/zbrafish
>

[Qiyan Mao]

Hi,

I've recently bleached my 48 h embryos before ISH according to Thisse's
protocol (Nature protocols (2008) 3(1):59) using 3% hydrogen peroxide in
0.5% KOH, and it worked fine for me. The whole bleaching takes around 20
minutes. My impression is that this bleaching step doesn't alter ISH
staining. I also tried post-ISH bleaching using the same procedure, but for
some reason the pigment doesn't go away as easily.

Hope this could help.

Best, Qiyan

On 8/20/08 12:03 PM, zbrafish...@oat.bio.indiana.edu wrote:

> Send Zbrafish mailing list submissions to
> zbra...@net.bio.net
>
> To subscribe or unsubscribe via the World Wide Web, visit
> http://www.bio.net/biomail/listinfo/zbrafish
> or, via email, send a message with subject or body 'help' to
> zbrafish...@net.bio.net
>
> You can reach the person managing the list at
> zbrafis...@net.bio.net
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Zbrafish digest..."
>
>
> Today's Topics:
>
> 1. Re: Bleaching embryos after In Situ Hybridization
> (non...@gmail.com)
> 2. Re: Bleaching embryos after In Situ Hybridization
> (non...@gmail.com)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 20 Aug 2008 09:49:09 -0700 (PDT)
> From: non...@gmail.com

> Subject: [Zbrafish] Re: Bleaching embryos after In Situ Hybridization

> To: bionet-organi...@moderators.isc.org
> Message-ID:
> <69b4622d-afa1-4d79...@f36g2000hsa.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> bleaching is a routine approach in frog embryo whole mount in situ
> hybridization, i think you could find the protocal and recipes in the
> Frog Book (Early Development of Xenopus Laevis: A Laboratory
> Manual)....
> And be aware the time of bleaching, hydrogen peroxide could breakdown

> the staining of in situ hybridization.
>
>

> low
>
>
>
> On Aug 19, 1:22 am, shellyleib...@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
>
>
>
>

> ------------------------------
>
> Message: 2
> Date: Wed, 20 Aug 2008 09:55:33 -0700 (PDT)
> From: non...@gmail.com

> Subject: [Zbrafish] Re: Bleaching embryos after In Situ Hybridization

> To: bionet-organi...@moderators.isc.org
> Message-ID:
> <59eb3d60-d642-4604...@z72g2000hsb.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1

>
> 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...@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
>
>
>
>

> ------------------------------

>
> _______________________________________________
> Zbrafish mailing list
> Zbra...@net.bio.net
> http://www.bio.net/biomail/listinfo/zbrafish
>

> End of Zbrafish Digest, Vol 39, Issue 10
> ****************************************