[Histonet] 20 micron sections

[Due, Brice]

Hello Cynthia, we cut thick (10-20um) paraffin brain here fairly often. Someone
else already mentioned that warmer is better. I find 10um cuts well at room
temp., however 20um cuts even better if I throw the block into the hot water
bath for a few seconds to soften it up. Or I soak a gauze in the hot water and
press it to the block face (in the chuck) periodically.

The temperature issue usually causes chatter/shatter artifacts. If you're
getting wrinkles on thick sections, I'm guessing it's from the swell on the
water bath. Your perfect flat 20um section becomes "rippled" when the grey &
white matters swell differentially on the hot water. Then it won't lay flat on
the slide. You can try increasing the temp of your water bath to spread the
section more. Or you can lower the temp alot to reduce the swell. Neither will
eliminate the problem all the time. 

Above 15um, it becomes more difficult to get good adhesion. Seemingly this is
because the thick sections trap water more effectively and/or don't lay flat on
the slide. Be sure you air dry the section *thoroughly* before melting them in a
hot oven. We usually put sections in a 37C oven overnight and melt at 60C the
next day.

To deal with the wrinkle/adhesion problem, here's a gem from the old-timer who
taught me. Place a few pieces of filter paper on a bench surface. Soak it with
some 95-100% ethanol. Not just damp, but soaking dripping wet. Cut your section
and pick it up off a hot water bath. It will not lay flat on the slide, but
strive for the flattest you can. Stand the slide up for 1-5min to drain some of
the water. Here's the trick. Now carefully lay the slide **section DOWN** onto
the wet filter paper. Hold the slide firmly in place and rub down the back side
hard. I was taught to rub in only one direction (pick one), but I've found that
rubbing randomly works just as well. Now *carefully* pick up the slide from the
paper. I've found that the easiest way to do this is to pull the filter paper to
the edge of the bench and pull the paper downwards. Magically the section stays
on the slide. If it sticks to the paper, use more ethanol before laying the
section down or use a clean paper on top. Change filter paper if paraffin
fragments stick, or every several slides.

This trick works well with 20um and thicker. For sections thinner than 15um I've
never needed it. Note, this will force/produce some wrinkles because of the
squishing effect, but most of the section will get flattened out onto the slide,
with good adhesion. We do this for large sections of brain (up to 6"x8").

If you can use 15um sections, they are much easier to cut and work with. They
flatten out better and have fewer drying issues. When I jump from 15um to 20um I
usually have to dig into my bag of tricks.

Best of Luck,
-brice
Neuropathology Lab
Brigham & Women's Hospital
Boston, MA


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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of cynthia
haynes
Sent: Wednesday, October 05, 2005 5:14 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] 20 micron sections


Hello, everyone I am in need of information. I am
cutting brain(human)tissue at 6 microns and 20
microns. The 6 microns sections are perfect. The 20
microns sections are an nightmare(and I mean Elm
street nightmare). Can anyone suggest the best way to
cut these sections without getting wrinkles and folds.
I am at my wits end.
Thanks in advance

Cynthia Haynes H.T. 

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