[Histonet] Phospho antibodies and fixation

[Jean-Martin Lapointe]

Hi Ashley,
I did a little bit of work on this a few years back. My belief is that the main difficulty with IHC for phospho-epitopes is not formalin fixation per se, but rather the instability of the phosphorylation. I used to work with rodent tumor models, where we collected solid tumors (or other tissues)  just after sacrifice, and fixed them by formalin immersion. We found that the periphery of the tissues fixed this way expressed various phospho-epitopes quite well, but not the center. My interpretation was that by the time formalin had penetrated to the deep regions of the tissue, the phosphorylation had gone away (as you know formalin penetrates solid tissues fairly slowly). We proceeded to test matched tissue samples, fixed either in formalin at room temperature vs. cold formalin (formalin was at 4C at immersion, and the tissue was put in the fridge after sampling). We found that the cold formalin samples expressed phosphorylation sites fairly evenly, compared to only peripheral with the room temp samples. Presumably the cold temperature preserves the phosphorylation sites long enough to leave time for formalin to penetrate throughout. Consequently we integrated cold formalin fixation to all our IHC protocols for phospho-epitopes.
Hope this helps,

Jean-Martin Lapointe
Accellab

-----Original Message-----

Good day colleagues,

Does anyone have any information on phospho antibodies and fixation?  Is
there any reason to NOT fix specimens in formalin vs say, 70% EtOH? (and
then process them through formalin later?)  I can't seem to find any
information on whether or not fixation in formalin does something strange to
a phosphorylated protien and makes it a less accessible antigen.  Also, on
that same note, does retreival do anything to it?

I am assuming that these antibodies go through the same testing for cross
reactivity, etc (depending on the vendor) and are reliable when used
properly (like any other antibody).  I don't, however, know if there is
enough of a difference in the epitope (the fact that it is phosphorylated)
that would make it more susceptible to some strange cross linking with
formalin (especiallly with phosphate buffered formalin).

Any help with this topic would be greatly appreciated as I am uneducated in
this area.

Thanks,

Ashley Troutman BS, HT(ASCP) QIHC
Vanderbilt University Histopathology
1301 Medical Center Drive TVC 4532
Nashville, TN  37232
615-343-9134
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End of Histonet Digest, Vol 77, Issue 21
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