[Histonet] Phospho antibodies and fixation

[Patsy Ruegg]

Ashley,

Contact me directly about phospho antibodies.  I have done a lot of studies
and used some different fixatives other than formalin, but the issue is
complicated.  One does not necessarily have control over whither or not you
are actually capturing the phosphorylation event or not.  There is a company
in my building called PhosphoSolutions that sells Phospho antibodies
exclusively, the owner is Mike Browning and he, others and I did a workshop
at the Denver NSH S/C on this subject.  I have some references and protocol
for fixatives I can provide you, but the fact of the matter is that it did
not seem to make any difference in the results if we used formalin over a
special fixative, what did make a difference is time to fixative, that must
be very well controlled and you should get the tissues into fixative asap.

Best regards,

Patsy 

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Troutman,
Kenneth A
Sent: Friday, April 16, 2010 1:43 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Phospho antibodies and fixation

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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