[Histonet] IHC on fish

[Tyrone Genade]

Hello,


> From: "Hardy, Cate" <chardy at csu.edu.au>
> Subject: [Histonet] IHC on fish
>
> Hi all;
>
> Has anyone performed IHC on fish. I have been asked to perform this using
> several of our routine antibodies that I use on mammals. My gut tells me it
> won't work but I am a lowly histotech and don't know anything. Seeking
> knowledge from learned tech's with experience in such matters
>
> Thanks
>

My entire thesis was about taking antibodies generated against mammalian
antigens and using them to stain equivalent structures in fish. In many
cases the results were as expected. I didn't modify the protocols in any
way. Simply use the same protocol you are using for the mammals and apply
them to the fish.

What did save me a lot of time was doing western blots first to test for
immunoreactivity and specificity. Some of my pretty micrographs had to be
discarded because on a western blot the antibody labeled multiple bands
irregardless of the antibody dilution.

If you know the immunogen sequences blast the various fish protein sequence
databases and see how well the antigen is conserved in various species. If
there is low similarity in the sequence then it isn't worth testing the
antibody. Again, confirm with a western blot or dot-blot for
immunoreactivity.

As regards fish sectioning the little bones can be a bother, and the
tissues seems to dry out and harden very fast in ethanol. I would suggest
something like Davidson's fixative, overnight at 4 oC and then quickly
process the tissue. For antigen retrieval you can modify the protocol of
http://www.ncbi.nlm.nih.gov/pubmed/21603650 for sections. You can also
try Citrate Buffer Antigen retrieval. If you are going to do
immunofluorescence then the protocol I got from Gayle Callis works well:
after taking water incubate slides or section twice in 300 mM glycine in
TBS, pH 7.4, 15 minutes at room temperature (RT) to quench autofluorescence.

Good luck with the antibodies!

-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org
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