John M. S. Bartlett.pdf - Bio-Nica.info

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digitally to record the dissection. Photocopying histological sections through an acetate
sheet avoids scratching the photocopier glass or smearing it with mounting medium.
5. A scalpel blade vertically in contact with the section will remove a narrow strip of tissue.
The blade at a flatter angle will remove a wider strip. Tissue so removed can often be rolled
into a small ball or pill, picked up on the tip of the scalpel blade or sterile hypodermic
needle and transferred to a microcentrifuge tube for further processing. Tissue is most
easily handled when damp but without excess water, in which dispersed tissue fragments
are hard to retrieve or too dry and brittle. Static electricity may be troublesome.
6. Bacteriological loop holders and tungsten wire are inexpensive. Either prepare enough
needles in advance to use a new needle for each microdissection if necessary or briefly
repolish the needle between samples, which exposes a new tungsten surface. The risk of
DNA carryover on the needle from one specimen to the next appears largely theoretical.
In a study of ras mutation in colorectal carcinomas (10), no false positives were detected
in the analysis of several hundred separate microdissected normal and tumour tissue
samples.
7. Any microscope can be used if there is enough space between objective and stage for
access to the specimen. Ordinary or inverted standard microscopes can be used but a
stereo dissecting microscope with relatively high maximum magnification (up to ×120)
is ideal.
8. If the specimen adheres to the inside of the pipet tip, it can sometimes be dislodged by
repeated in drawing and expulsion of buffer. It may be picked out with a needle, or the
pipet tip may be cut off and placed with the tissue fragment in the tube for subsequent
digestion. Sticking can usually be avoided by coating pipet tips before use with a silicone
such as Sigmacote. Draw (e.g., 50 µL) of Sigmacote into the tip, expel it again, shake
off any excess, and allow to dry. Coat tips an hour or two before you need them. Collect
microdissected fragments for further processing in 500-µL tubes with screw-on lids sealed
with rubber O rings. The O ring prevents loss of specimen volume by evaporation during
subsequent processing and 500-µL tubes fit most PCR thermal cycling blocks for heating
to inactivate Proteinase K after the digestion process is complete
9. Capture volumes of 12.5 or 25 µL work well. The pipet should be nearly vertical to
minimize the risk of damaging the section. Use a Gilson-type pipet with a wide-bore
polypropylene tip. Such tips are in many manufacturer’s catalogs, or you can cut the end
off an ordinary pipet tip. Steady your hand, holding the pipet, against your fist resting on
the microscope stage. The fragment may be hard to retrieve if it lies flat on the section or
slide. Briskly expelling buffer toward the fragment will usually lift the fragment to a level
from which it may easily be recovered. Retrieval of dissected fragments without damage
to the section is easier from a deep pool of buffer. The buffer should not contain detergent,
such as Triton X-100 which, by reducing surface tension, prevents the formation of a
deep standing pool of buffer.
10. Labeling must survive overnight incubation in the water bath and subsequent heat
inactivation in the PCR thermal cycler block, the wells of which often contain oil traces
which may dissolve even permanent marker pen inks. Write specimen numbers on the lid
of the container as well. Typing correction fluid on the lid gives a white surface on which
graphite pencil is permanent. Do not transpose lids. Do not open more than one specimen
tube at a time, and replace the lid at once.
References
1. Schriever F., Freeman G., and Nadler L. M. (1991) Follicular dendritic cells contain a
unique gene repertoire demonstrated by single-cell polymerase chain reaction. Blood
77, 787–791.