[Histonet] [ SOLVED ][ Histonet ] Strange circles in IHC slides

[histonet.nospam <@t> vneubert.com]

Hello Histonet,

it has been a while (~10 months) since I posted a problem about uneven immuno-staining with specimen showing unstained circles after manual staining with HRP-polymere/DAB method; complete mail see below, response mails see Histonet archive (via website).
Links to pictures I took:
http://img12.imageshack.us/img12/8513/ts0402162049.jpg
http://img13.imageshack.us/img13/6514/ts0402162104.jpg

The problem occured suddenly, without having changed any reagents or methods.
Things I changed to avoid the unstained spots:
*Adding 0,05% Tween 20 to TBS
*Blocking peroxidase in coplin jar, not mounted in racks
*Lowering antibody concentration
which temporarily produced better results.


After reviewing a big number of slides it showed up that most of the tissue affected was lung, liver and kidney which mostly means a lot of blood in the tissue when fixation in formalin starts.
Erythrocytes, granulocytes and macrophages show a lot of endogenous and pseudoendogenous peroxidase activity.

This is how it's done since then:
Slides are taken from racks into a big coplin jar with 3% H2O2 diluted in distilled water (demineralized H2O).
A slow magnetic stirrer on the bottom of the jar keeps the solution floating around the tissue, removing any O2 bubble that might appear. Slides then are remounted and rinsed a lot with TBS-T.

Thank you for all your help, though it's a little late...

V. Neubert,
Germany





----- Original Message -----
From: histonet.nospam <@t> vneubert.com
To: histonet <@t> lists.utsouthwestern.edu
Date: 02.04.2009 18:12:18
Subject: [Histonet] Strange circles in IHC slides


[...]> http://img12.imageshack.us/img12/8513/ts0402162049.jpg
> http://img13.imageshack.us/img13/6514/ts0402162104.jpg
[...]> 
> So, has ever anyone experienced sth. like this?
> My conjugate control (every step except the antibody) was fine, nothing 
> to be seen about DAB and no circles at all.
> 
> I used Shandon single-use coverplates, sterile buffer, fresh antibody 
> aliquots. Any idea?
> 
> Thanks,
> 
> V. Neubert