10/28/99

I am working with the ultrastruture of anthers of Ilex paraguariensis.

However, during the dehydration of the samples, fixed in a mixture of glutaraldehyde 2,5%

and formaldehyde 2%, the anthers shrink, mainly after the secondary fixation with osmium

tetroxide. I used acetone or ethanol , in steps of 30, 50, 70, 90, 90, 100, 100,

with 15 minutes in each step, at room temperature. The anthers has a very reduced

dimension (about 1 mm length) and shrink in the step 100.

What to do? Should I to use a low temperature (4oC)?

Thanks in advance.

Rinaldo Pires dos Santos

rinaldop@botanica.ufrgs.br

I think you need to check the strength of the fixative buffer. In any

event, it is much better to cut the anthers in half across their long

axis immediately after they are immersed in the primary fixative to

allow the fixative into the anther locule. ASlso, fix for a long time in

thye primary fixative

Patrick Echlin

Cambridge UK

Dr P. Echlin

pe13@cus.cam.ac.uk

are relatively difficult to fix and embed (especially when they are

approching maturity) even when other organs and tissues are well preserved.

If adjusting buffer strength and cutting them transversely as Dr. Echlin

suggests doesn't do the trick, my suggestion would be that you try much

longer dehydration times. I suggest at least an hour at each dehydration

step and perhaps leave them overnight at the 70% ethanol stage. Of course

the trade-off might be that you extract some of the stuff you wish to

preserve. You will have to adjust your protocol on a species to species

basis. John

________________________

C. John Runions, Ph.D.

Section of Ecology and Systematics

Corson Hall

Cornell University

Ithaca, New York

USA 14853

email cjr14@cornell.edu

phone (607) 254-4282

Fax (607) 255-8088

Prolonged dehydration and embedding will probably do the trick for

you. However, I found that the loss of lipid material during a long

dehydration protocol is unsatisfactory. Instead I use acidified DMP

(Di-methoxy-propane) for chemical dehydration. For small objects,

like anthers, complete dehydration can occur in 15 min or less, but I

would use one hour at room temp. for a start. DMP can be mixed with

all hydrofobic embedding media I know. However DMP dehydration can

sometimes reslut in shrinked material as well, but I would give it a

try.

Bo

Bo Johansen

boj@bot.ku.dk

Something we and others have experimented with is the use of

Dimethylsulfoxide (DMSO). It acts as a cell penetrant and stabilizes the

cytoskeleton. My work is principally with human neutrophils but others have

used it for yeast cells (and other things, but this should be enough to test

the idea.)

References:

Gilbert, CS and RT Parmley Morphology of human neutrophils: A comparison

of cyrofixation, routine glutaraldehyde fixation, and the effects of

dimethyl sulfoxide Anat Rec 252:254-263 (1998)

Fassel, TA; Sohnle, PG and VM Kushnaryov The use of dimethylsulfoxide for

fixation of yeasts for electron microscopy Biotechnic & Histochemistry

72(5):268-272 (1997)

Forgive my Portuguese, it's little rusty. I can help with acquiring copies

of the references if you need them.

Ciao,

Chuck

Charles Gilbert

cgilbert@carolinas.org