[Histonet] IHC Staining Problem

[Jennifer Hill]

 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