[Zbrafish] Re: IHC/HE Assistance

Lasse via zbrafish%40net.bio.net (by lasse from scienceshow.dk)
Tue Dec 16 04:27:26 EST 2008

On Dec 15, 8:55 pm, "Michal Galus" <galus.mic... from gmail.com> wrote:
> Hello,
> I have recently finished an exposure where we have prepared adult
> zebrafish for IHC/HE whole mount slides. We will be subjecting the
> samples to serial longitudinal sections and we are attempting to find
> a protocol to decalcify the fish for sectioning. I am inquiring if
> anyone is aware of any protocols or would be able to point me in the
> right direction of where I would be able to find some.
> Thank you for your time,
> Sincerely
> Mike

HI Mike

I have a bit experience with IHC on adult tissues. I usually fix whole
adult fish with their bellies opened, or smaller sections in 4% PFA at
4 degrees overnigh, then decalcify for one week at RT in EDTA prior to
dissection/finer sectioning. I have also decalcified in acid (formic
acid: citric acid; 1:1) for 24 hours, but this will degrade RNA if you
need to do combined ISH/IHC. I know that some protocols refix after
decalcification, some do dot decalcify at all and others decalcify for
longer time (>1 month in EDTA). My protocol is not good if endogenous
flourescent proteins needs to be identified - especially dsRed is no
longer appearent after 1 week at RT, but the tissues are much easier
to finely cut into sections compared to other decalcification

Hope this helps :). Feel free to contact me off-list if you like, and
I can send you more detailed protocols :). lasse.jensen from ki.se.

Sincerely: -Lasse

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