[Zbrafish] Re: In situs on larval stage fish

Jill Sakai jasakai at wisc.edu
Wed Aug 9 07:29:28 EST 2006


Hi Adrian,
I use 3% hydrogen peroxide + 1% KOH in dH20 to bleach embryos and
larvae after fixation and before moving them to MeOH to start in situ
hybridization.  If you fix your embryos in epp tubes you can pull out
the fix and add the bleaching solution directly, no rinsing required.
Watch the reaction to decide how long to bleach them - I've found it
takes about 10-15' for 36 hpf to about 45' for 5 dpf larvae.  Mix the
solution fresh and leave the caps of the epp tubes open because the
reaction produces lots of bubbles.  I usually check partway through to
make sure the bubbles don't push any embryos out of the top of the
solution.  You'll be able to see if some aren't bleaching.  Once
they're done to your liking, remove the bleaching solution and rinse a
couple of times with PBS, then wash them into the methanol.  It works
very well to remove pigment and I think I've used it on larvae up to 7
dpf.

Hope this helps - let me know if you have any questions!
Jill

-----
Jill Sakai
Neuroscience Training Program
University of Wisconsin-Madison
Madison, WI  53706
lab: 608-262-9223



Adrian Grimes wrote:
> Hi!
> Does anyone have a protocol for removing pigment on later stage larvae for
> in situ hybridizations? Can I use hydrogen peroxide and, if so, at what
> point in a standard in-situ protocol should I attempt this? I don't want to
> use PTU as it may complicate the toxicology study I am doing.
> Thanks,
> Adrian Grimes
>
> "Nothing is more common than the erroneous belief that one is displaying
> judgment or taste by showing an unwillingness to be pleased"  Samuel Johnson
> (1709 - 1784)
> --------------------------------------------------------------------
> Adrian Grimes
> PhD Candidate
> c/o The Kirby Lab
> Neonatal Perinatal Research Institute
> Box 3179 Bell Building
> Duke University Medical Center
> Durham
> NC 27710
> 
> Tel: (919) 668-2311
> Fax: (919) 668-1599



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