Re[2]: Uneven Keratin Staining

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Date:Fri, 25 Jun 1999 22:39 -0400 (EDT)

Try the MNF116 cytokeratin from Dako for skin's. Our dermpath attendings like a cocktail of AE1/AE3, MNF116 and CAM5.2.
The MNF116 works well after digesting with 0.25% pepsin, pH 2.0.


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Subject: Re: Uneven Keratin Staining
Author: at internet
Date:    6/25/99 4:23 PM


I, too, have problems from time to time with uneven cytokeratin signal.  I
use DAKO's Z0622, which is a polyclonal wide spectrum antibody.

I would say that the problem is remedied with fresh antibody (until
recently I've not been keeping alloquoits in the -80 but only in the 4
deg.) but I've recently seen the phenomenon with other antibodies.

I've experimented with various detergents, no detergent, paraformaldehyde
fixed tissue, Bouin's fixed tissue, standing on my head and etc.

I don't know yet.  I'm starting an assay tomorrow with fresh antibody so I
can let you know sunday what fresh antibody does, at the least.

More later

John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849

On Fri, 25 Jun 1999, Richard Maliniak wrote:

> We are currently experiencing uneven...areas of Positive and areas of
> Negative immunostaining on skin....with our DAKO keratin AE1/AE3.
> Protocol: primary antibody at 1:100 for 30 min at room temp, pepsin
> pretreat(4mg pepsin/ml buffer, incubate 37C for 30 minutes),LSAB+
> detector system(30 minutes at room temp)on DAKO autoimmunostainer.
> Our skins are formalin fixed for an assorted amount of times. Any
> suggestions?
> Thanks in Advance.

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