[Histonet] RE: Phospho antibodies

[Thurby, Christina]

Ashley,
When we have worked with phospho antibodies, we have used the following pre-treatment to induce de-phosphorylation of the tissue sections:

1. _ Deparaffinize slides to distilled water.
2. _ Perform De-phosphorylation step. (Add 400 uL of alkaline phosphatase (Sigma Type VII-L) to 600 uL of 0.01 M PBS). Incubate for 2 hours at 32°C.  Rinse well with distilled water.
3. _ _ PBS, 2-5 min.
Proceed with your laboratory IHC staining protocol.  (We use the ABC method as a standard).  What antibody are you planning to use?  I have a few protocols available for animal tissue on the Dako Autostainer.

We fixed in either 10% NBF for routine necropsy specimens or PLP fixative for perfusion.

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>Date: Fri, 16 Apr 2010 14:43:15 -0500
>From: "Troutman, Kenneth A" <kenneth.a.troutman <@t> Vanderbilt.Edu>
>Subject: [Histonet] Phospho antibodies and fixation
>To: "Histonet <@t> lists.utsouthwestern.edu"
>       <Histonet <@t> lists.utsouthwestern.edu>
>Message-ID:<7B310892042DA74CB3590053F424CFE60B7480EFBC <@t> ITS-
>HCWNEM06.ds.Vanderbilt.edu>
>       Content-Type: text/plain; charset="iso-8859-1"
>Does anyone have any information on phospho antibodies and fixation?...
>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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