Re: Elution of antibodies??

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>Does anyone have a procedure for the elution of antibodies from tissue
>sections? Seems I've seen it before on Histonet but I can't find it.


I don't know about the protocols previously published on the Histonet, but
I've had to deal with elution of antibodies several times. I've tried about
every protocol published in the litterature and have had several surprises.
Some antibodies are very difficult to elute from sections, even if you use
very harsh treatments. Some extreme pH elution techniques actually seem to
denature antibodies in situ, which then precipitate, rather than being
eluted. The only neverfail technique I know is the electrophoretic elution
technique by Vandesande, described in the following reference:

Vandesande F. (1988) Double and multiple immunoenzymatic labelling of
tissue sections for light
microscopy. Acta Histochem Suppl. 35:107-15

It's a rather complicated setup, requiring electrophoresis equipment. For
the last years, I have been using a modified acid pH elution technique,
which worked fine for me in ICC and Western blotting. I use the classical
100 mM pH 2,2 glycine buffer, supplemented with 0,1 to 0,5 % Tween 20 and
50 mM beta-mercaptoethanol. I add the beta-mercaptoethanol to disrupt the
disulfur links between the light and the heavy chains of the
immunoglobulins to ensure complete elution of the antibodies. This
treatment does not seem to interfere with most subsequent ICC detections,
but his should be verified for each antibody.

I proceed as follows:

After the primary detection, rinse the sections once in the glycine elution
buffer (see above) and then treat 3 x 20 minutes with the same buffer.
Afterwards, rinse at least 5 times 2 min with PBS or TBS. It is essential
to get rid of all the beta-mercaptoethanol, or subsequent antibodies risk
to be reduced by the beta-mercapto. As sson as there is no faint
beta-mercapto smell, you can start the second ICC labelling starting with
the blocking step.

You should include a control to check whether the elution does affect the
second immunolabeling. Also include control to check for complete elution
of your first sequence of antibodies up to the first primary ! Simply
reapply your detection system on the slides to see whether there is still

Elution techniques for double labeling can solve many problems especially
if you deal with primary antibodies produced in the same species. However
there are several pitfalls, especially with enymatic labels. DAB and
several other peroxidase chromogens seem to cloak antigenic sites. Thus a
first ICC detected with DAB may hide the second antigen, if located in the
same cells !! Controls are a major factor in these procedures and can
quickly add up to 3 to 5 additional slides per actual sample.

Have fun

Paul Klosen, Ph.D.
CNRS UMR 7518 Neurobiologie des Rythmes
Universite Louis Pasteur, Strasbourg, FRANCE

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