[Histonet] antibodies against phosphorylated proteins

[Heike Grabsch]

Thanks Andrea. The problem is that I have no control at all about
fixation of the tissues. I m aworking with human tissues from a
pathology archive. 

Interestingly, you are talking about controls. Apart from negative
controls - it is difficult to identifiy a positive one, as this will
also be fixed tissue - or do you have a good suggestion for this?
Heike 

> -----Original Message-----
> From: Andrea T. Hooper [mailto:anh2006 <@t> med.cornell.edu]
> Sent: 31 May 2006 17:07
> To: Heike Grabsch; Histonet
> Subject: Re: [Histonet] antibodies against phosphorylated proteins
> 
> 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
> 
> --