Help with wholemount Immuno

[PZ Myers]

In article <8t270j$9iv$1 at mercury.hgmp.mrc.ac.uk>, Juliette 
Faraco <faraco at cmgm.stanford.edu> wrote:

> I'm looking to find a contact that can help offer advice on 
> how to do whole mount immuno on week-old zebrafish.  The 
> experts  in the Talbot lab here on campus can't offer much 
> advice on larvae that are that old, since they primarily work 
> with much 0-24 hour fish.  I really need some help from 
> someone that has experience with  week old zebrafish!  
> 
> My laboratory is just starting to use zebrafish. We're trying 
> to study a neuropeptide that is in the mammalian tuberal 
> hypothalamus and also has been cloned and characterized in 
> Xenopus (very conserved). We've heard another investigator has 
> stained slices of carp brain with mammalian antibodies so 
> although we haven't cloned our gene yet, we believe it's 
> present in the fish.  
> 
> I want to study the course of development and expression in 
> zebrafish using whole mounts so that we can develop a 
> technique for screening of mutants. I've tried three times 
> now, and have lots of trouble, particularly with the ABC 
> peroxidase step.  I've read and tried to use several different 
> methods, including the detailed and useful method by Rachel 
> Macdonald in the Methods in  Dev Bio series.  
> 
> I permeabilize them using -20 degree acetone and do the 
> incubations with antibodies overnight at 4 degrees (2-3 nights 
> for primary, one night for secondary). Washes are extensive 
> (at least all day in 5 washes, sometimes over night too)  and 
> the buffer is PBS + 0.8% triton.  Despite using an 
> avidin/biotin blocking kit  (vector labs) as well as a 30 
> minute quench in methanol/peroxide, I think there is some big 
> problem with non-specific peroxidases. At the end of my 
> protocol after incubation with ABC complex and extensive 
> washing, I transfer the embryos to DAB (without peroxide) to 
> pre-incubate. At this step, flocculant debris forms in the DAB 
> solution. After transfering to DAB with peroxide, the embryos 
> go dark very quickly, (1-3 minutes) and I don't see evidence 
> that any staining has the chance to occur within the brain 
> rather than on the skin.  When I removed some embryos and 
> didn't expose them to the ABC reagent, the flocculent debris 
> did not form.  I don't know what this huge systematic problem 
> is, but I can't proceed.  
> 
> I don't know what else to try for the wholemount protocol and 
> I need it to work, so please offer your advice.  

I can't help you, I'm afraid. I've never had any luck with larval 
wholemounts -- the tissue is just too thick and dense, and I 
could never get good penetration. Have you considered vibratome 
sections? Cutting a 5-7 day larva into 50 micron sections is 
fairly quick and painless, and is the only way I got good 
staining at those ages.

-- 
PZ Myers