11/14/97

The method suggests sections of Epon (a soft mix is preferred). It will even

preserve antigenicity of tissue as well as excellent structure. Instead of

the old sodium methoxide that chews up the tissue, the sodium is held by

18-crown-6 ether (Aldrich Chem. #16,665-1) to produce something with better

properties. I will be glad to write out my notes but I think it is all in

the following references (and you will see examples of the results that I

can't provide). I got information on this attending an Integrated Microscopy

workshop at the IMR in Madison a couple of years ago.

In a nutshell, you prepare:

30% (w/w) methanolic potassium methoxide (dry methanol over 3A molecular sieve)

Stock solution:

18-crown-6 1.3g

DMSO 99ml

water 1 ml

To make the working solution, mix in proportion of 100 ml stock soln. and 3

ml of the sodium methoxide (30% w/w). The mixed working solution loses

activity rapidly so only mix what you need. The notes I have say that a 5

min treatment removes all the resin from a 200 nm section.

Polysciences sells an epoxy removal kit based on this mixture in case you

don't want to get the components and mix yourself, but it is pretty simple.

References:

For the preparation of the extraction medium:

Idaware, Harada, Yoshino, and Arai, 1990, Stain Tech. 65, 205

Ultrastructure:

H. Ris and M. Malecki, 1993. High-resolution field-emission scanning

electron microscope imaging of internal cell structures after epon

extraction from sections: a new approach to correlative ultrastructural and

immunocytochemical studies. J. Struct. Biol. 111, 148-157.

Sincerely,

Dale A. Callaham

+++++++++++++++++++++++++++++++++++

Dale A. Callaham

Senior Microscopist

Central Microscopy Facility

The University of Massachusetts

Amherst, MA 01007

-------------------------

Phone 413-545-3751

Fax 413-545-3243

email dac@bio.umass.edu