[Histonet] Histonet Digest, Vol 179, Issue 5

[Amos Brooks]

Hi,

>      I have a hunch that the DAB deposits you are seeing are not a DAB
problem. You didn't mention how long your incubation time is. If I were to
venture a guess, I am thinking there may be some evaporation of either the
primary or secondary antibody or boil-over from the antigen retrieval. This
would give the DAB a place do react. You could use a plastic coverslip or
really rinse the slides a lot.
     I really have not seen any problems with an oven interfering with IHC.
I think it is superstition. However, I have seen problems with microwaves.
Do yourself a favor and chuck it off the roof of your lab. They do not
provide control over temperature. Some areas heat faster than others. They
tend to boil over and are always harsh on sensitive tissues like bone. You
would be much better off with a hot water bath. It will solve all of these
problems.

Just my tuppence,
Amos

Message: 7
Date: Fri, 5 Oct 2018 14:02:33 +0000
From: "Wilson, Lindsay A" <Lindsay.A.Wilson at uth.tmc.edu>
To: "histonet at lists.utsouthwestern.edu"
        <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] DAB & tissue adherence issues on rabbit bone
        tissue
Message-ID: <2F7418A2-D147-4D69-97B0-DB6CFA1C6DFB at uth.tmc.edu>
Content-Type: text/plain; charset="utf-8"

Good morning,

I am currently experiencing an issue with DAB concentrating on certain bone
areas of my slide. I am working with decalcified?EDTA , FFPE- rabbit
mandible (some tissue attached). It is a new DAB kit from BioCare. It?s
thoroughly mixed (and new) before application in a humidity chamber with
the slides lying flat.

I change out the deparaffinization soluitons frequently, my reagents are in
date, I have increased the peroxidase time to 10 minutes, I use a blocking
reagent from Millipore kit  for 5 minutes (manuf. Recomm.), 2.5% goat serum
for 20%, my wash buffer is 1x TBST with 0.05% Tween 20.

The FFPE slides are aired dried before use. I have seen posts about baking
them in the oven afterward ? that doesn?t affect the slides at all?

I also have the issue of the sample falling off and/or moving on my slide.
The slides are positively charged, I am currently performing AR with an AR
ph6 buffer (for VEGF antibody) in 95C microwave for 15 minutes. I have
previously tried a 60C waterbath for 12 hours with the same issue.

I am thinking that the issues are connected? If the tissue isn?t moving
around or falling off, perhaps the excessive DAB wouldn?t be an issue? I
have seen posts about the Biogenex DeCal solution ? is that an effective
method?

I can attach images if needed.

Lindsay Wilson, LVT, RLATG
Young Laboratory