RE: Re: What are vibratome sections used for?
At 23:58 24/03/02 -0500, vous avez écrit:
>Can you really get decent EM from frozen sections embedded in plastic?
>What sort of cryoprotection do you do?
I used the procedure described by L.-I. Larsson in Vol.1 of Methods in
Chemical Neuroanatomy (pp. 195-196.
After fixation, the tissues are cryoprotected in a series of sucrose
solutions (10%, 12% and 15% for 2 hours and finally overnight in 20%
sucrose + 5% glycerol + 40 microM digitonin). All sucrose solutions are
prepared in 100 mM phosphate buffer pH 7.4. The digitonin is supposed to
improve membrane structure, but I have never checked whether membrane
structure is impaired if I leave the digitonin out.
It is essential for EM work to use tissue slices that do not exceed 1 cm in
at least one dimension. Ideally, they should be about 5 mm thick. The
cryoprotected slices are then frozen in isopentane cooled to -60°C with
liquid nitrogen. The -60°C seem to be important, because if frozen at
higher or lower temperatures, after sectioning the floating sections curl
up, and it becomes impossible to unroll them to obtain even immunolabelling
throughout the section.
For preembedding EM work, even on vibratome sections, you will have to use
some permeabilization agent if you want your antibodies to pentrate more
than less than 1 micrometer. Some people use detergents, while some even
suggest freeze-thaw cycles after cryoprotection the sections in DMSO. You
may also use buffered ethanol (10%, 15% and 20% in 100 mM Phosphate buffer
pH adjusted to 7,4). Permeabilize the sections for 5-10 min in each of
these (going up and down). The ultrastructure of membranes is affected
though less than one might expect and antibodies penetrate almost througout
the section. This technique works best when combined with an alcaline
You may look up a paper by Eldred et.al. (1983) in JHC 31(2):285-92.
Another trick to improve EM structure with preembedding work is to postfix
the sections for 1 hour with GLUTARALDEHYDE after immunostaining and BEFORE
peroxidase detection. Glutaraldehyde only slightly affects peroxidase
activity. For peroxidase tract tracing, only glutaraldehyde works better
than glutaraldehyde/formaldehyde fixatives. This postfixation with
glutaraldehyde stabilizes ultrastructure, which otherwise will be impaired
by the free radicals liberated during the peroxidase reaction. This can be
seen on heavily labelled structures in preembedding EM ICC.
The combination of the above techniques allows the production of more than
decent EM structure in floating frozen sections. Some of the
permeabilization techniques are also usefull in vibratome sections.
Especially the buffered ethanol procedure if you want to do only optical
work on vibratome sections. I never found an antigen which would not work
after this procedure it it worked with detergent permeabilization. And
compared with detergents, this procedure is much more efficient and allow
complete penetration of the immunoreagents throughout the sections (with 10
min in each ethanol).
I hope this answers some of your questions.
(o -) O
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. 03.90.24.05.01 Fax. 03.90.24.05.28
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