Help with wholemount Immuno

[Juliette Faraco]

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.  

thanks in advance,
Juliette Faraco