[Histonet] Frozen rat brains sections: OCT adheres, tissue does not

[Dan Barkmeier]

Thanks for the replies I've gotten thus far!
I do find that warmer temperatures make it less likely that the tissue
section will detach from the surrounding OCT, so that makes that problem
less troublesome.  What remains my main frustration now is the fact that *the
rat brain sections show no particular attraction to the slide*.  The OCT
adheres wonderfully - both quickly and smoothly, but the tissue itself is
very slow to attach and hardly seems attracted to the slide at all, even
when I'm angling the slide downward.

Does anyone have experience with a similar issue?  I'm wondering if more or
less fixation might have something to do with this?

In response to some questions asked:
 - Yes, I do let the block equilibrate in the cryostat for about an hour
before sectioning. I've even tried putting it in the -20 freezer overnight
to make sure it's really up to temperature
 - No I have not tried snap-freezing fresh tissue.  Would better results be
expected from this?  Would there be any problems using these sections for
riboprobe in situ hybridizations or immunostaining for phosphorylated
proteins?
 - My cryostat does not have separate object/rollbar temperature settings.
Per some advice I found in the archive, I placed aluminum foil containers
with dry ice near the blade and the anti-roll plate to keep them colder.
This allowed me to cut at a warmer temperature than I previously had, but
only up to about -14C, where the section scrunches up anyway.


I have tried getting out a new box of slides from a newly-delivered batch to
see if that made a difference, but it did not.  I've also borrowed some
hand-subbed slides from another lab to see if that helped the tissue
attraction, but it didn't seem to make a difference.
This problem has been variable - some brains work well, others seem
impossible.  Any insight into what variables to watch out for would be
appreciated!

 - Dan





On Tue, Apr 15, 2008 at 7:35 PM, Katie Glattfelder <
KatieG <@t> alleninstitute.org> wrote:

> I cut fresh frozen mouse brains at 25 microns for about two years and we
> frequently encountered the same issue. There are a couple of things you
> can try:
>
> 1) try warming up your temperature a little bit - we usually found that
> an object temp of -11 to -13 and a chamber temp of -14 to -16 worked
> best
> 2) try cooling your rollplate immediately before taking a section,
> sometimes this can reduce the separation but often has to be done
> frequently
> 3) if you haven't already, try trimming your OCT fairly close (1-2 mm?)
> at the edge that touches the blade first and try to catch the first part
> of the section at the same time or close to the same time as the OCT,
> then tilt the slide down quickly to get the whole section without
> wrinkles or folds (it's hard to describe a technique with words!)
> 4) try using Neg-50 instead of OCT.  Neg-50 greatly reduced tissue
> separating from the embedding media but takes some getting used to
> because it "behaves" differently than OCT.  If you use NEG 50, you
> should reduce your temperatures by 2-3 degrees from what I recommended
> above, and probably have some practice tissue to get used to it.
>
> Hope this helps!
>
> Katie
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Dan
> Barkmeier
> Sent: Tuesday, April 15, 2008 11:09 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Frozen rat brains sections: OCT adheres, tissue does
> not
>
> I've experienced ongoing frustrations with cryosectioning rat brains for
> over a year now, and felt it was time to seek professional help.  I'm
> having
> trouble getting good quality sections, which seems to stem from a
> varying
> mix of these two related problems:
>
> 1) Upon cutting, parts of the tissue section detach from the surrounding
> OCT
> embedding medium
>
> 2) When adhering the section to a slide, the OCT sticks immediately, but
> the
> section is not very adherent.  The causes the perimeter of OCT to 'run'
> around the section well before the neighboring tissue is stuck, and I
> get
> giant wrinkles in the section.
>
> Tissue processing:
>  - Quickly dissect brains, cut them into 2-5mm coronal slices, and place
> them in 4% paraformaldehyde in PBS for 48 hours
>  - Cryoprotect in 30% sucrose in PBS until tissue sinks (1-2 days)
>  - Embed in OCT on dry ice
>  - Cut sections at 20um (I've tried 10um as well, with similar results),
> and
> at various temperatures from -17C to -22C, and mount on SuperFrost Plus
> slides
>
> I don't really have problems cutting human brain samples prepared in the
> same manner; it's just my rats that give me trouble.
>
> I would greatly appreciate any advice offered.  I've searched through
> the
> archives as best I can, but I did not find these problems specifically
> addressed.
>
>  - Dan Barkmeier
> Wayne State University School of Medicine
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