[Histonet] antibodies against phosphorylated proteins

[Andrea T. Hooper]

I have found as long as the tissue is fixed rapidly that you can 
easily detect phospho proteins in FFPE tissue. I have found that heat 
retrieval is key ... but I also have realized that providing you are 
using a reliable technique that the quality of staining is only as 
"good" as the antibody - as in all immunohistochemistry.

I used to use a special fixative that contained sodium orthovanadate 
and some other ingredients to inhibit phosphatases but truthfully 
that caused more problems than it solved as the tissue was often not 
fixed well and embedding ended up being a nightmare.

I recommend 10% NBF and just remember to process the tissues after 
removal from the organism as fast as possible. And as always remember 
to include controls :)

Good luck!
Andrea


>I am working with formalin fixed paraffin embedded tissue and trying to
>detect phosphorylated proteins by immunohistochemistry. The antibodies
>are meant to be specific for the phosphorylated protein only.
>The one antibody I am trying to work up in particular is the
>phospho-H2AX from Upstate.
>Is there anybody out there who has an idea whether and if then how
>fixation may effect phosphorylation sides? Can they be lost or destroyed
>due to fixation? So the staining could be false negative?
>I hope somebody can comment on this
>
>Thanks,
>Heike
>
>Dr Heike Grabsch, MRCPath MD
>Gastrointestinal Research Group
>Pathology and Tumour Biology
>JIF Building, Level 4, Room 4.13
>Leeds Institute for Molecular Medicine
>St James's University Hospital
>Beckett Street
>Leeds
>LS9 7TF
>United Kingdom
>
>phone 0044 113 3438626
>fax    0044 113 3438431

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