190
VOLKER JUNG and GEORG A. WEBER
Microdissection of cells from slides
1. Prepare enough microcentrifuge tubes for each experiment.
Note: Three tubes are needed as controls; one for a reagent blank, one
for a sham microdissection and one for a positive control.
2. UV irradiate the glass needles and the 0.2-ml thin-walled microcentrifuge tubes containing 6 III cell collection buffer with 360 mJ/cm
2
to
remove possible extraneous DNA contamination.
3. Cautiously place a glass needle into the micromanipulator with tweezers.
4. Microdissect 20-50 cells from an area of interest.
5. Transfer the cells which adhere to the glass-needle into a microtube
containing 6 III collection buffer.
Note: All experimenters develop their own "style". The best results of a
microdissection come from a tissue which is neither too humid nor too
dry. Be careful with the transfer of the microdissected tissue into the
collection buffer, the tips may also be broken, but it is important for
the following procedures that the needle tip is within the solution.
6. For microdissection of each area ofinterest, place a new needle into the
micromanipulator.
Note: For a control, touch the needle onto an empty place of the slide
(sham microdissection) and transfer the sham dissection to a blank
tube. This will serve as a control for any contamination introduced
during the microdissection.
7. Overlay with 15-20 III sterile mineral oil for PCR to avoid evaporation
of water (facultative).
8. Incubate cells at 55°C for 4 h to digest cellular proteins and release
DNA.
9. Heat to 95°C for 15 min to inactivate proteinase-K.