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

From:"Katie Glattfelder"

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
2) try cooling your rollplate immediately before taking a section,
sometimes this can reduce the separation but often has to be done
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!


-----Original Message-----
[] On Behalf Of Dan
Sent: Tuesday, April 15, 2008 11:09 AM
Subject: [Histonet] Frozen rat brains sections: OCT adheres, tissue does

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
trouble getting good quality sections, which seems to stem from a
mix of these two related problems:

1) Upon cutting, parts of the tissue section detach from the surrounding
embedding medium

2) When adhering the section to a slide, the OCT sticks immediately, but
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
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),
at various temperatures from -17C to -22C, and mount on SuperFrost Plus

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
archives as best I can, but I did not find these problems specifically

 - Dan Barkmeier
Wayne State University School of Medicine
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