Histonetters,

Please help!  I have talked to Mequita Praet, who so graciously reminded me
of all you wonderful people and that
surely someone out there has already encountered this problem!!!  Special
thanks to her for her help!!!

We are trying a new technique called Laser Capture Microdissection or LCM.
This consists of a fancy microscope
called the PixCell  II that has the ability to laser zap single cells off
of frozen tissue sections. We need to zap about
1000 cells per sample.  The cells then adhere to a plastic cap that is then
put on an eppendorf tube containing lysis
buffer to isolate the RNA.  The RNA prep is pretty cookbook.  My problem
lies with the frozen sections.

The human tissue has to be obtained from the Operating Room.  The fresher
the better so the RNA doesn't degrade.
So I go to the O.R. with my bottle of OCT, plastic cryomolds, and a small
vat of liquid nitrogen.  The surgeon
removes the tissue, gives it to me,  I cut it in little pieces, put it in
the mold, and drop it in the liquid nitrogen.  When
I get back to the lab, I put the cryo blocks in the -80 C freezer to
preserve the RNA.  The protocol calls for the
blocks to stay at -80 as much as possible so the RNA doesn't degrade.
Obviously, I can't cut them at -80C as
they are extremely brittle.  PROBLEM NUMBER 1:  How long does it
approximately take for the blocks to
come from -80 C to -20C for cutting purposes?

Because of the way the microscope slide fits on the microscope, you can
only put one section in the exact
middle of the slide. As often happens, the sections are rolling up as soon
as they are cut and I'm having a
rough time "teasing" them out flat.  The protocol recommends non-coated
slides but coated slides can be
used.  We are using Super Plus slides but are still having a horrible time
with the sections washing.
PROBLEM NUMBER 2:  Not only are they washing totally off, but the edges are
constantly flapping over
on themselves.  Since we are trying to zap epidermal cells, this is
creating a big problem not to mention
a big waste of time.  I am cutting about 25 slides at a time but only maybe
one or two slides are useful.
Immediately after cutting, we are doing a quick stain H & E so the only
fixation is 70% ETOH.  Again,
this is the recommended protocol.  We are eventually wanting to do a quick
IHC stain so we can isolate
proliferating cells but we can't even seem to get pass the basic frozen
sectioning.  The "recommended
protocol"  includes every organ except skin.  So for most samples, if the
edges flap, there is still plenty of
"usable" tissue in the middle.

Again, the protocol recommends (this "protocol" is recommended from the
company "Arcturus" - who
manufactures the microscope and promotes this LCM procedure) that the
quicker the tissue is removed
from the source to the end result of RNA isolation - the better and that
the tissue should stay at -80 as much
as possible.

I am desperate for any suggestions or thoughts,

Nancy Cardwell
Research Assistant III
Vanderbilt University Medical Center
Department of Plastic Surgery Research
Nashville, TN  37232-6904     USA
615-322-7266 phone
615-343-2050 fax