Re: HELP - frozen sections for RNA isolation

From:Greg Dobbin <dobbin@Upei.CA>

I don't have any experience with LCM,  but some with cryostat and 
H & E. Here are some ideas to toss around:
	What if you arrange to cut the frozens right outside the O.R. 
You can place the tissue right on the mounts , freeze, cut in no time 
(figuratively speaking of course!) flat. Then let the sections dry on 
the slide as long as your protocol will allow (i.e. rather than picking 
up the section and going immediately to fixative, let it air dry for 10 
or 15 secs, to maximize adherence). Then fix,(your fixative could be 
held at -20 if you prefer, in a nearby freezer or in the cryotome 
cabinet [not ideal]). Then if the Rapid H & E (should have a slide 
stained in 3-4 mins) is not fast enough, there is another cryostat 
stain for "extreme" emergencies, the Single Step Polychrome 
Methylene Blue method (slide stained in under 2 mins.). Gives dark 
blue nuclei with other elements varying shades of violet to pink.
I am not clear as to whether the sections for LCM are the stained 
ones or adjacent unstained sections? The unstained fixed sections 
can then be kept at Liq. N2 temps if you wish. And the left over 
tissue on the mounts in the cryotome can be sealed with a little 
OCT, plucked off the mounts and stored in sealed plastic bags in 
-80 C indefinitely. In this way, you tissues are moving just one way 
down the temperature scale: fresh, to -20, to -80 (with a short visit 
to -196 C, if transported in liq. N2).

	As for the sections rolling up: are you using an anti-roll plate, 
anti-roll wire or (my favorite) the good old camel hair brush? I find 
the plate works really well, when it works! By that, I mean there is a 
very exact setting for the plate to work, and when you find that 
setting life is beautiful, but finding it can be a pain! I like the brush 
because once you get the hang of it, it's like riding a bike (as they 
say). All I can offer is, if there is not enough OCT below the tissue, it 
is more difficult to keep the section flat with either method.

That's all I have. Hope it helps. Greg
> 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

Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 Unviversity Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851

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