|From:||Miriam Schroeder <firstname.lastname@example.org>|
Hi Cheryl. I have the third edition of Theory &
Practice of Histological Techniques, edited by
Bancroft & Steven. (1992 re-print)
The book has an entire chapter on autoradiography.
(Just in case you happen to have access to this book.)
I found ONE paragraph in the chapter entitled
"sectioning, mounting sections, and de-waxing". Here
is what is has to say:
"Sections are cut in the conventional manner and
mounted on 'subbed' slides. It is essential to make
quite certain that they are thoroughly de-waxed before
preparing autoradiographs, and to this end, it is
vitally important to use fresh organic solvents (e.g.
xylene) only, otherwise traces of wax will remain in
the surface of the slide and impair adhesion of the
emulsion to the section. When the tissue is embedded
in resin (e.g. Epon, Araldite) there is no need to
remove the embedding medium; the film may be applied
directly to the sections."
There is also a section on "The choice of a suitable
fixative", too long for me to recopy but here are some
"In general, the fixative must (a)preserve the tissue
structure and its stainability; (b) prevent the loss
and/or diffusion of radioactive material from the
tissue; and (c) have no appreciable effect on the
sensitivity of the auradiographic emulsion." "Mercury
or lead-containing fixative should be avoided..." "In
some instances the labelled material of interest may
not be fixable in the ordinary sense. Labelled
inorganic ions (e.g. [22Na]) may be impossible to fix
in the conventional way and certain small molecular
weight organic molecules (e.g. glucose) may also fall
into this category. In such a case the tissue is
rapidly frozen and autoradiographs are prepared from
frozen sections of material using Appleton's
modification of the stripping film technique..."
(Miriam's note - this modification amounts to reacting
the film in a freezer, the tissue is never allowed to
"thaw".) "There is no universal rule of thumb which
can be followed, and the ultimate choice of a suitable
fixative is a matter for the individual to decide,
taking into consideration the chemical nature of the
labelled material that he or she is dealing with, and
the degree of structural preservation which can be
regarded as acceptable."
ANOTHER question for you: will the tissues already be
"radioactive" when you receive them? My co-workers
were once planning an autoradiography study (which we
never got around to doing after all) and I know they
were going to react the tissue sections with the
radioactive material AFTER the sections were cut. But
the Bancroft and Steven chapter sounds like the animal
is usually injected with some sort of radioactive
material BEFORE the study, and thus the tissue is
radioactive when you receive it - ??? Anyway, just
might be something you need to think about also. I
never work with radioactivity myself, but I seem to
recall hearing that tritium can't be detected with a
Geiger counter, that you have to do wipe surveys - ???
Good luck. Hope this was of some use to you.
--- Cheryl Crowder <email@example.com>
> Hi - I need some help. A researcher here wants to
> do autoradiography on
> mouse tissue. He wants them paraffin embedded, cut
> and then the slides
> given to him for the technique. I have never done
> any tissue for
> autoradio-graphy before.
> Could someone tell me how thick the sections should
> be cut (they are
> tagged with small amount of tritiium)? Are they put
> on regular, clean
> slides or do you use charged or coated slides?
> I don't have any information this and need it by
> tomorrow. Any help will
> be really appreciated.
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