RE: Frozen sectioning
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|From:||Alan Bright <email@example.com>|
|To:||'Gayle Callis' <firstname.lastname@example.org>, 'Scott McDonald' <email@example.com>, firstname.lastname@example.org|
Looking through your comments below I picked up on the problem you have with
sectioning brain above 30 um, below is a comment I have posted in the past
to Histonet and should assist in overcoming your problems:-
Our Model OTF/AS Brain Specification, would allow sections of brain up to
300 um, as it is fitted with an
independent specimen temperature control, the specimen would need to be set
between -8 & -12 deg. C, the
microtome chamber at -20 to -25 deg. C , with these setting the brain will
serial section very easily and the brain will not crack or compress.
If you do not have specimen temperature control, you will need to set the
microtome chamber to -8 to -12 deg. C, and try to lay solid C02 on the knife
and anti-roll plate, this is due to the fact that brain will stick to the
knife and anti-roll plate at its ideal sectioning temperature and will need
to be cooled to stop this. The reason you do not achieve good sections is
that the brain is to cold for good quality sections.
Please come back to me if you are in need of some more information on this
Bright Instrument Co.Ltd.
St Margaret's Way
Tel No:+44 (0)1480 454528
Fax No:+44 (0)1480 456031
Web Site: www.brightinstruments.com
From: Gayle Callis [mailto:email@example.com]
Sent: 29 June 2000 15:46
To: Scott McDonald; firstname.lastname@example.org
Subject: Re: Frozen sectioning
You did not say if your specimen was tissue or some other substance. We
were cutting alginate for certain biofilm evaluation.
If possible, embed in a frozen tissue (OCT, Tissue Tek) or try Shandons,
Fishers, etc). OCT has the ingredients on the bottle, polyvinyl alcohol and
polyethylene glycol, but they do not give the molecular weights of these
We prefer the OCT for its sectioning qualities, strictly our personal
preference, and never use saline to embed, frozen water doesn't cut well!
and the OCT supports the tissue nicely. The alginate specimen was
surrounded with some sucrose and had been soaked in same to cryoprotect.
What little was there, didn't interfere with sectioning overly, but it
takes some dicey handling to get the section from the knife to the specimen
holder which then goes to a freeze drying, etc before SIMS evaluation.
Are you trying to do SIMS evaluation?? If so, there are some
considerations here. Have worked out a technic which has been providing
some results they want, no contamination from PVA/PEG. I cut at 30 um,
thicker was a disaster, the sample was not cutting well, wanted to break
apart. OCT DID not surround the sample. Sample has to be thick enough to
sit on top of OCT (hold specimen to the chuck only) so the section came off
without any OCT around it. The section could NOT thaw onto the chip, it
had to be placed on this surface in frozen state requiring a clean, fine
tip forceps, #3 for EM type forceps, cleaned with acetone.
If I tried to cut thicker, the sample was 1) too thin a specimen and one
bumped into the OCT underneath the sample 2) section was a disaster. My
cryostat can't cut a 80 um thick section, and others may be limited on how
thick you can cut also (micrometer setting limitations). I think thicker
could be cut with manipulation of cryostat temperature, not too cold or
section fractures, etc IF the sample was a bit thicker, over 2 mm thick.
If your sample is that thin overall, you cannot trim into a block face
without going through what you may want to see, 80 um for our alginate, we
would b through the block in about 3 cuts! Whew!
In developing the technic to get section from knife to silicon chip, felt I
was standing on my head! and did shut lab door to not offend my neighbor
lab with expletive deleted comments!
I don't know if this helps, but if possible please elaborate more on type
of sample and evaluation being done.
At 03:40 PM 7/5/00 +1000, you wrote:
> I have been asked to do some frozen sections for a research
>project. These specimens need to be frozen into a block , preferably with
>the least amount of organic matter involved. Would embedding them in a
>saline solution pose any problems ? If this has been done before by anyone,
>what concentration have you used/problems did you face? Would anyone be
>to recommend other solutions to use? The routine solution being used in the
>lab I have access to , is OTC. Does anyone know the chemical makeup of this
>solution (if it is available to the public)?
> Also , I have never done any cutting greater than 7um and I have been
>to cut these at about 80um . Are there any major problems associated with
> Any help at all with this situation will be greatfully appreciated.
>BlackBurn Bld D06
>University of Sydney
>ph: [61 (02)] 93516171 or 93517569
>fax: [61 (02)] 93514105
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
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