[Drosophila] cryosections for laser capture microscopy
(by ktokuyasu from ucsd.edu)
Mon Jun 22 16:51:53 EST 2009
Dear Dr. Anna Hitrik,
Dan forwarded your mail to me. Let me describe my opinion on your
First, I would like to point out that components of O.C.T. are
polymers, which means that it may fill in intercellular spaces but not
penetrate into the cell interiors through intact cell membrane.
Hence, when the ovary is frozen, ice crystals will be formed inside
the cells, damaging the cellular structures, and as the sections are
thawed, artificial spaces will be formed in the cytoplasm.
Secondly, as you know, cell structures are more difficult to recognize
in thicker sections than in thin ones, mainly due to super-positions.
This will be particularly true in unstained sections.
If cryosections thinner than 7 micron thickness are OK or preferable,
there is a way to obtain 0.1-2 micron thick cryosections rather
readily. The cutting quality of thin cryosections is much improved by
infusing a sucrose solution into cells (Tokuyasu, K.T. 1973. A
technique for ultracryotomy of cell suspensions and tissues. J. Cell
A formaldehyde-fixed Drosophila ovary may be infused with 1.5-2.3M
sucrose in an appropriate buffer, frozen by plunging the specimen into
liquid nitrogen and sectioned at -70~ -90oC. If your cryo-microtome
is not suitable for cutting such thin cryosections, you may try to
find a nearby EM lab equipped with an ultra-cryomicrotome and carrying
out immunostaining of ultrathin cryosections. Such lab will know how
to retrieve cryosections onto glass slides and remove sucrose from the
You may then have a fair chance to see cell boundaries in thin, thawed
cryosections, with phase-contrast LM or other means.
If your project requires to see much finer structural details, I would
propose a new approach; all operations involving the laser capture
microscopy are done first to obtain a data map and then the
cryosection is stained to obtain a morphological map. Using an
appropriate coordinate, one may combine the two maps to obtain the
whole map. Such an approach could also be used for your 7 micron
thick section, if necessary.
I would hope that these suggestions were useful to you.
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