[Histonet] Re: double staining in frozen tissue

From:"Johnson, Teri"


Answers to your questions:

1)Would it cause problem if I use FITC donkey
anti-goat IgG and Alexa 647 goat anti-rabbit together
as my secondary antibody?

Yes, it would cause a lot of problems because the FITC anti-goat
secondary would bind with the goat anti-rabbit.  I would use donkey-anti
rabbit instead. I recommend anybody doing multiple staining methods
purchase Chris van der Loos' book.  You can find it on Amazon.com...it's
an excellent reference for these types of questions and problems.

2)Which fixation is better? I had tried 4%
fornaldehyde (at room temp) for 5 min and icy acetone
(-20) for5 min, and found with acetone-fixation, the
morphology of nucleus is very poor. Any suggestion?

It depends on what fixation does to the epitope you're trying to
visualize. Formalin can cause a lot of autofluorescence and can, in some
cases, mask the antigenicity.  In other cases, it provides excellent
fixation to the proteins you're staining, and of course provides
excellent morphology. Try it and see if it works.

Many antibodies are successfully used on acetone-fixed cryosections.
However you have found the limitation of using acetone alone.  Do a
search on the histonet (www.histosearch.com/histonet) and look for Gayle
Callis' recipe and instructions on using acetone and absolute alcohol
mixture.  In many cases, it retains antigenicity and preserves
morphology very well.  Do not let the slides air dry after
acetone/alcohol fixation, but rather go into your buffer solution and
continue your protocol from there.

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133

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