Fixation time for IHC?
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,
Lovelace Respiratory Research Institute
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