Re: Antibody pre-absorbtion and Methyl Green

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From:"L. Gibbs" <>
To:Liz Sabin <>
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Hi Liz-

Sounds like you do have a problem with contaminating
antibodies. See Jules Elias' book: "Immunopathology, A Practical
Approach to Diagnosis", 1990/ASCP Press. He mentions the
following reference: J Clin Pathol 41: 705, 1988.

Frankly, I have never had great luck with preabsorbing
antibodies. Sometimes we just accept these "natural" artifacts if
they do not interfere with our label.

Methyl Green:

I use 2% Methyl Green prepared in 0.1M Acetate buffer, pH 4.2.
I have never had it fade after mounting; however, I am not using
DPX either.

Good luck!

Lorraine Gibbs 

On Wed, 29 Mar 2000, Liz Sabin wrote:

> Hi all,
> I have a couple of questions:
> First, I have a rabbit polyclonal antibody which I believe is 
> producing inappropriate staining in my human skin paraffin sections 
> due to a cross reaction with epidermal keratins.  Specific staining 
> is clearly visible where it should be and in my positive control, but 
> I'm getting quite a strong background "blush" uniformly throughout 
> the epidermis which I would like to remove.   The dermis is spared.  
> Has anyone had any experience in preabsorbing polyclonal primary 
> antibodies to remove this cross reaction, specifically preabsorbing 
> the antibody with human keratin? Any general tips and pointers 
> would be appreciated.
> My second problem concerns Methyl Green. I use the VectorLabs 
> stain and follow their protocol, heating the stain to 60oC and 
> incubating the slides for 1-5 minutes, followed by rinsing in 
> deionized water and dipping in acetone/0.05% acetic acid.  The 
> staining is rather lighter than I would like but acceptable.  The 
> problem comes with the mounting.  I'm routinely using a permanent 
> xylene based mount, DePeX from BDH.  This works fine with slides 
> stained with haematoxylin.  However, on contact with the sections 
> that have been stained with Methyl Green, they completely 
> disintegrate!
> I know I could try other mountants, but I would like to know why 
> this only seems to occur with Methyl Green.
> Thanks in advance for any help and also thanks to all those who 
> responded to my last question concerning antibody precipitation.  
> A combination of spinning the ab for 2 min on max in a benchtop 
> microfuge and syringe filtering the chromagen worked a treat.
> Liz Sabin.

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