Fixation time for IHC?

From:"Allen, Steven"

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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