Re: Fixation time for IHC?

From:"John C. Dennis"


I think the place to begin is have you, in the past, obtained good results
with the
current antibodies on these same tissues.  I routinely fix tissue for 48
hours.  I've found that poor results stem from many sources (the antibody
itself to my batch of normal serum having gone bad during storage and
everything between) some of which are my doing or not doing and some
beyond my control and ken.

Are these antibodies that you've used before?

John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849

On Tue, 24 Jun 2003, Allen, Steven wrote:

> Good morning,
> I am doing some IHC on rat tissues (multiple antibodies-one at a time).  Due
> to poor results with several antibodies, I have been speaking with technical
> support and they have recommended a formalin fixation time of anywhere
> between 2 and 12 hours for tissues intended for future IHC.
> Our necropsy unit usually waits a minimum of 24 to 48 hours before trimming
> and embedding.  I cannot go back and undo the fixation time for my tissues,
> I have to use them.  I realize the problem of cross-linking with extended
> formalin fixation.  I am performing antigen retrieval: 0.01M citrate (pH
> 6.0), 10 min. microwave boil.  I am also performing a 0.16% trypsin digest
> (30 min) before the blocking step.
> I have obtained positive results with other antibodies, using the same
> tissues.  Also, my tissue blocks are about 4 years old.
> Could/should I extend the boil time for antigen retrieval?  What kind of
> fixation times do you use for IHC tissues?
> Thanks in advance,
> Steve Allen
> Research Technologist
> Lovelace Respiratory Research Institute
> Albuquerque, NM

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