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|From:||Roger Moretz <email@example.com>|
|To:||"BrosnanWatters, Gayle" <GBrosnanWatters@vanguard.edu>, 'histonet' <firstname.lastname@example.org>|
Well, I wouldn't guarantee that using the heat pen
will mean you would never lose another section, but it
really ought to significantly reduce those losses.
Once the section is nicely spread on the knife bath,
the possibility of wrinkling on the slide can be
reduced by using a shorter heating time on the water
drop. Further, you need to monitor the
drying/adhering/flattening process carefully, and blot
off excess water once the section has flattened and
adhered. I have used the old Corning hot plate,
setting the control to between one and two. The
initial drying/flattening is done with the
slide/section area toward the center of the hotplate.
Once blotted, the slide is moved to the edge of the
hotplate where the temperature is lower. (In case
this sounds very artsy, let me tell you that I am
convinced, after nearly 30 years of this, that
microscopy techniques more ofter verge on magic--be
that black or white, take your choice--than on
Yes, diamond knives are expensive. However, there are
knives that are sold specifically for thick
sectioning. These knives are simply not sharpened to
the extent required for ultrathin sectioning. All of
the vendors (to my knowledge) sell such knives. I
have used them from Diatome, DDK and Microstar. I
think that Drukker also sells them. I have to check
this out when I get back in the lab, and I will pass
along the info then. As with any other fine or
delicate equipment, diamond knives require extra care,
attention to detail and occasional, very, very careful
cleaning. I have had knives last for years. And, if
the edge dulls, they can be resharpened for
significantly less than a new knife. I will look up
the details and e-mail you after the holiday weekend.
Roger Moretz, Ph.D.
Dept of Toxicology
Boehringer Ingelheim Pharmaceuticals
--- "BrosnanWatters, Gayle"
> Wow, I might have to actually learn something new!
> A heat pen, huh?
> Does it work on relatively "thick" sections such as
> 1 micron sections, and
> on huge ones like a whole mouse brain (I have a
> specially machined chuck
> that fits into an MT2 so I can cut a whole one)?
> Nothing makes me madder
> than to have exactly the section I need, and then,
> in spite of having done
> it thousands of times, losing that section because I
> overheated the water on
> the hot plate! This pen would prevent that?
> And while I'm at it, I have always been afraid of
> diamond knives
> because they told me they don't last and that they
> cost so much I probably
> couldn't keep myself in them - what's this about a
> "thick section" diamond
> knife? Where would I get details on that?
> This is so cool; I can ask questions of really
> knowledgable people
> without feeling too much like an idiot!!
> The psychologist
> > ----------
> > From: Roger Moretz
> > Sent: Friday, February 18, 2000 8:40 PM
> > To: Elizaha@aol.com; email@example.com
> > Subject: Re: araldite
> > I am rather surprised to see the old toluene (or
> > carbon tet, or xylene--you name it, it's been
> > technique still being used. And I used it for
> > and years....what was I saying??? Oh yes. Can't
> > remember what causes those memory lapses....
> > Anyway, seriously. Several of the EM supply
> > sell a little "Heat Pen" unit that does a
> > job of flattening sections on the water in the
> > boat. I switched to one of those over 15 years
> > and at least exposure to solvent fumes at the
> > ultramicrotome no longer contribute to neuronal
> > necrosis!!! I don't mean to slight anyone, (I'm
> > home and don't have the catalogs at hand), but I
> > know that Ted Pella carries the heat pen.
> > As to knife angle, etc. The usual 6 degrees
> > work, and glass knives should last if treated
> > However, if you can afford it, the "thick section"
> > diamond knife will speed things up a lot.
> > Finally, heating the slide/water/section is
> > but can be frustrating if you haven't done it
> > I use a similar technique to those mentioned
> > but preheat the slide and drop of water briefly
> > to placing the section on the water surface. You
> > have to practice to figure out the best heating
> > temperature and drain/drying protocol for your
> > and the size of the section. I have done this for
> > block sizes that were nearly the whole embedding
> > capsule diameter (coronal sections of half a mouse
> > brain).
> > Hope all of our responses will help.
> > Roger Moretz, Ph.D.
> > Dept. of Toxicology
> > Boehringer Ingelheim Pharmaceuticals
> > --- Elizaha@aol.com wrote:
> > > In addition to the heating all ready mentioned,
> > > can also use an
> > > applicator stick that the tip has been
> > > in toluene and wave it
> > > closely over/above the section ....either while
> > > in your boat or still
> > > floating on water on the hot plate...this
> > > the section even more.
> > > Don't touch the water or the section....
> > >
> > > Elizabeth
> > >
> > >
> > __________________________________________________
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