[Histonet] Immuno. confusion

Amos Brooks amosbrooks <@t> gmail.com
Wed Aug 3 17:45:08 CDT 2011

    Well... almost any point before DAB. You wouldn't want to put it in
after the HRP conjugated secondary because that would quench the HRP signal
you are actually looking for leaving you nothing for the DAB to precipitate
on. On the other hand you certainly do not *need* to do it after the HIER as
it will work just fine before it or even after the primary antibody
(assuming it isn't HRP conjugated). Actually we have had the best results on
CD4 if the H2O2 is done prior to the HIER. The H2O2 in this case actually
affects the epitope if you do it after HIER. If it does that with CD4, I
assume it is likely to do so with other finicky epitopes.

Have a nice day,

On Wed, Aug 3, 2011 at 1:00 PM,
<histonet-request <@t> lists.utsouthwestern.edu>wrote:

> Message: 7
> Date: Wed, 3 Aug 2011 11:49:40 -0500
> From: "Troutman, Kenneth A" <Ashley.Troutman <@t> Vanderbilt.Edu>
> Subject: [Histonet] Immuno. confusion
> To: "Histonet <@t> lists.utsouthwestern.edu"
>        <Histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
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> 7B310892042DA74CB3590053F424CFE6143EE301B7 <@t> ITS-HCWNEM06.ds.Vanderbilt.edu>
> Content-Type: text/plain; charset="us-ascii"
> Hi Valerie,
> The peroxidase blocking step is to prevent endogenous peroxidase activity
> that will cause a false positive stemming from your application of DAB (the
> substrate for your DAB solution is hydrogen peroxide).  You can essentially
> put this blocking step at any point in your stain (following
> deparaffinization) and before your application of DAB.  I have run this step
> at varying points in my procedures and they all work fine.  Some antibodies
> prefer you do this step before primary application and others prior to DAB,
> but for most stains, it doesn't matter too much.
> As for an enhancer, you can make a copper sulfate solution (5mg/mL in
> buffer).  Or you can try DAB Enhancer or DAB Sparkle from Biocare.
> Good luck,
> Ashley Troutman BS, HT(ASCP) QIHC
> Immunohistochemistry Supervisor
> Vanderbilt University Histopathology
> 1301 Medical Center Drive TVC 4531
> Nashville, TN  37232

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