Re: Antibody pre-absorbtion and Methyl Green
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From: | "L. Gibbs" <lgibbs@u.washington.edu> |
To: | Liz Sabin <esabin@srv4.med.ed.ac.uk> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
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.
>
>
>
> esabin@srv4.med.ed.ac.uk
>
>
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