At 14:52 30/10/00 -0700, Sharon Bledsoe a écrit:
>May I please have all the pros, cons and random thoughts of using 
>carbowax  as an embedding media.  Horror stories and success are equally 
>welcome.

Pro:
Morphological quality, immunoreactivity
I my hands, almost all antibodies that work on aldehyde-fixed tissue cut 
frozen or on the vibratome, can be used with similar or superior results on=20
PEG sections. We also use PEG for ISH where it is superior to paraffin, 
both in morphological quality and sensitivity. PEG sections are perfect for=20
ISH/ICC double labeling. We perform first the DIG-ISH and then select 
sections for further ICC, sometimes even months after the original ISH has=20
been performed and detected. The slides are stored in buffer in the fridge.
PEG sections can be stained as floating sections down to ... 8 microns, and=20
even sometimes 6, depending on the tissue. Routine is 10-12 microns as 
floating.
I also could adapt most classical histological stains for PEG sections. The=20
main difference with paraffin is a somewhat "mushy" appearance of nuclear 
stains.

Cons:
Sectioning, handling and mounting of the sections. If you work in a humid 
area, forget about PEG, unless you have a well climatized room with good 
ambient humidity control. When working with PEG, humidity is a constant 
enemy. Summertime is no friend for PEG people.
Also, PEG sections are notoriously hard to keep on slides, even with 
poly-L-lysine or +charged slides. Furthermore, you have to be aware that 
upon mounting, the PEG will instantly dissolve. For brain tissue, or other=20
coherent tissues, this is no problem and will ensure very nicely spread 
sections. I've cut and mounted complete 10 micron sagital sections of PIG 
brains with no problem. But if you work on tissues like pancreas .... you 
usually end up with all the acini dissociated and spread over the mounting=20
bath. There are several double-embedding techniques or mounting techniques=20
that allow the mounting of these sections but they require and awful lot of=20
time and patience.

Conclusion

Over the years, we have improved our protocols to work with PEG throughout=20
the year and we have adapted the technique to most of our needs. But every=20
now and then I run into a new problem, I curse, sometimes even go back to 
cutting paraffin sections for ... well ... about 1 day. And I end up again=20
with PEG after solving my problem.
PEG cuts like paraffin, without the bad surprises. No static electricity 
problems. Perfectly spread sections. I've always had problems with 
glycogen-loaded livers in paraffin (too hard, sections falling to pieces).=20
The same tissue (same animal, same fixation, same processing) embedded in 
PEG cuts perfectly.
Thus ... well we like PEG, but there are some serious problems with the 
technique (depending on the tissue and the environment you are working in)=20
that you should be aware of.

Paul
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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=======