[Histonet] IHC Staining Problem

Jennifer MacDonald JMacDonald <@t> mtsac.edu
Sat Oct 30 00:44:58 CDT 2010

How far in advance are your controls being cut?  Are your patient tissues 

Jennifer Hill <jhill <@t> vet.k-state.edu> 
Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
10/29/2010 01:11 PM

histonet <histonet <@t> lists.utsouthwestern.edu>

[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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