RE: Dewaxing slides

From:Paul Klosen <klosen@neurochem.u-strasbg.fr> (by way of histonet)

Hi Louise,

I have been using Polyethyleneglycol embedding for over a decade now for
ICC, ISH and conventional staining. This paraffin-like matrix is however
water-soluble. Thus when we mount these sections on slides, you are left
immediately with "dewaxed" sections. From the first uses of PEG we noted
that if we keep these "dewaxed" sections dry for more than a few hours, we
lose ALL immunoreactivities !! And they have become perfectly irretrievable
by any means (microwaves, proteases, etc.). Thus we store these sections in
buffer at 4C until we use them. We have stored such sections for up to two
years in buffer without appreciable loss in immunostaining. This of course
requires the inclusing of a azide or some other agents in the buffer to
prevent the growth of bugs.
Note that if we keep the sections unmounted as ribbons, we can store them
dry (obviously, as PEG is hydrophilic) for up to 5 years, without any loss
of immunoreactivity. To prevent loss of immunoreactivity during the 1/2 to
2 hours of drying necessary to keep the sections sticking to the slides, we
include 2% sucrose in the mounting solution. The sucrose seems to protect
immunoreactivity in dry, dewaxed sections for some hours.

Thus from my experience, NEVER leave your sections dewaxed and dry for more
than a few minutes and avoid this situation at all cost. But the buffer
soak overnight, or even a few days should not affect the immunostaining,
provided the tissue is adequately fixed. Anyway, in some immunostaining
procedures, the sections are left in buffer for almost a week !

Paul Klosen

>Hi histonetters
>
>Has anyone had any experience with loss of antigenicity in slides that have
>been dewaxed and either left soaking in water/buffer overnight or allowed to
>airdry?
>
>The reason I ask this is that we are moving to an automated IHC sytem in the
>near future and will be dealing with a lot of slides and a short turn around
>time.
>
>Any input appreciated
>
>Louise Taylor
>Research Laboratory
>Department of Anatomical Pathology
>South African Institute for Medical Research
>Johannesburg
>South Africa

                                                                         -=3D-
                                                                        (o -) O
==========================3D=====oOo==(_)==OOo=============3D================
Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.88.35.85.04  Fax. 03.88.24.04.61
========================klosen@neurochem.u-strasbg.fr==================3D=======


<< Previous Message | Next Message >>