[Histonet] IHC Staining Problem

[Reynolds,Donna M]

>
>We had a similar problem a few years back. We had used an antibody at 1:500 dilution for years. Got in a new bottle and no labeling. Called the manufacture and they assured me it was the same concentration.  Finally went to 1:50 dilution and the staining was like our old stain.  Over the last few years with each new vial I titer and we are now up to 1:200 dilution. So I suspect it is your antibody.  When the problem began I still had enough of the old vial to run them simultaneously and the old vial was working fine. 
> ------------------------------
>
> Message: 10
> Date: Fri, 29 Oct 2010 22:44:58 -0700
> From: Jennifer MacDonald <JMacDonald <@t> mtsac.edu>
> Subject: Re: [Histonet] IHC Staining Problem
> To: Jennifer Hill <jhill <@t> vet.k-state.edu>
> Cc: histonet <histonet <@t> lists.utsouthwestern.edu>,
>    histonet-bounces <@t> lists.utsouthwestern.edu
> Message-ID:
>    <OF83C059C1.879738A5-ON882577CC.001F8665-882577CC.001F9C5A <@t> mtsac.edu 
> >
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>
> How far in advance are your controls being cut?  Are your patient  
> tissues
> staining?
>
>
>
>
> Jennifer Hill <jhill <@t> vet.k-state.edu>
> Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
> 10/29/2010 01:11 PM
>
> To
> histonet <histonet <@t> lists.utsouthwestern.edu>
> cc
>
> Subject
> [Histonet] IHC Staining Problem
>
>
>
>
>
>
> I hope someone can help me with a problem I've been having with my  
> CD79a
> hand stain.  This is a stain that was validated and working perfectly,
> when a few months ago I started having reduced, to no staining on my
> controls, with the occasional run staining a little stronger. I  
> followed
> the discussion not too long ago with the person who was having similar
> problems, but the suggestions didn't seem to apply (I use Tween 20  
> in my
> PBS rinses, and we use APEX slides from Surgipath-not Fischer  
> Plus).  I've
> tried everything I can think of and I hope someone may have some other
> ideas.  For reference this is for a veterinary diagnostic lab, not a  
> human
> lab, and this is the only stain we've been having this issue with.
>
> My protocol is as follows: After deparfinization/rehydration,
> AR for 20 min in the steamer in Biogenex Citra Solution (I've gotten  
> new
> Citra Solution, tried a pressure cooker)
> 5 min peroxide quench, followed by a 10 min protein block using Dako's
> Protein Block-Serum Free
> 60 min Primary incubation (Dako CD79a) at 37 C (decreased dilution  
> from
> 1:100 to 1:65, new bottle of antibody)
> 30 min incubation at room temp of ImmPRESS anti-Mouse Reagent from  
> Vector
> Develop stain with DAB chromogen from Vector
>
> Washes of PBS/Tween 20 follow between steps
>
> Thank you,
> Jennifer Hill
> Research Assistant
> Kansas State University
> Veterinary Diagnostic Lab
>
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> End of Histonet Digest, Vol 83, Issue 46
> ****************************************



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