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

[Rene J Buesa]

To me it seems that the sections after being picked from the water bath were not completely drained and the dewaxing process was incomplete in a way that the "round" areas kept certain amount of paraffin wax that prevented the reagents reactions.
The fact that the areas are round are an indication that water was involved, since water always leave a round imprint, due to its surface tension.
I would suggest that you dewax the sections with a 2% aq. solution of dish washer soap.
Dewaxing with xylene sections containing water will be incomplete because it does not mix completely with water but the detergent will mix with the water and will better remove the paraffin.René J. 

 


--- On Thu, 2/25/10, histonet.nospam <@t> vneubert.com <histonet.nospam <@t> vneubert.com> wrote:


From: histonet.nospam <@t> vneubert.com <histonet.nospam <@t> vneubert.com>
Subject:>[Histonet] [ SOLVED ][ Histonet ] Strange circles in IHC slides
To: histonet <@t> lists.utsouthwestern.edu
Date: Thursday, February 25, 2010, 8:34 AM


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




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