[Histonet] IHC Staining Variations

[Jan Shivers]

Occasionally in the past I'd have a problem with slides drying out while on 
the stainer.  Suggestions:

1)  Make sure that your stainer is level, especially the slide racks inside, 
if they are used in your type of machine.
2)  Dilute your antibodies with a diluent which has protein stabilizers in 
it; the Ab/diluent won't dry out as fast.  (I switched to Dako Antibody 
Diluent from my own in-house TBS buffer, but I'm sure there are other 
commercial products out there as well.).
3)  Add 2% normal serum to your linking Ab (same species as your protein 
block prior to the primary antibody); this has several uses, but one is that 
the linking Ab won't dry out as fast.
4)  Try reducing your incubation times, if possible.
5)  Hydrophobic elements in tissue will tend to 'push' water off of the 
tissue, so some specific tissues are just more likely to dry out faster.  If 
it's at all possible with your staining set-up, you could try adding 
additional reagent drops by hand (at the correct step, of course) when you 
notice a slide drying out in your machine.

Jan Shivers
Senior Scientist
Histology/IHC/EM Section Head
University of Minnesota
Veterinary Diagnostic Laboratory
shive003 <@t> umn.edu


----- Original Message ----- 
From: <histobeach <@t> aol.com>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, April 17, 2008 1:29 PM
Subject: [Histonet] IHC Staining Variations


> Hello,
>
> We have an automated IHC stainer and the doctors are experiencing what 
> they call, staining variations.? The whole tissue is not staining, just in 
> parts, when areas of the tissues should be staining.?
>
> Has anyone experienced this problem and if so, what did you do to remedy 
> it?? We were staining manually and used a Pap Pen before we switched to 
> automation and I'm wondering if we are getting areas on the slide that are 
> not being covered with the reagents.? We have 3 drop zones with 100ul of 
> reagent dropping in each zone, for a total of 300ul per slide.
>
> I appreciate any feedback and help.?
>
> Trinity Whitecomb
> Histology Manager
> Lab System Works
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