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

38 Going
pipet filler. Spread the pool of buffer until it extends 3 to 4 mm beyond all edges of
the section and is as deep as possible without spillage.
3. Center the area of cells in the section to be retrieved for subsequent analysis in the
microscope field at an appropriate magnification.
4. Using the coarse motion controls of the micromanipulator, place the needle over the area
to be dissected.
5. Lower the needle gently at the edge of the area to be dissected until its tip just touches
the slide. Stop lowering the needle when a small lateral deflection of the needle tip occurs
(see Note 8).
6. Microdissection techniques vary for different specimens. In general, attempt by blunt
dissection to develop cleavage between groups of cells. Work around the area to be
dissected, developing a split between the area to be kept and the area to be removed (Fig. 1).
Then, use the point of the needle gradually to undermine the area to be recovered, pushing
and pulling with the tip and side of the needle until the area to be retrieved has been peeled
from the slide and floats freely in the buffer pool.
7. If it is not clear whether the fragment is still attached to the section, gently agitate the
slide. Attached fragments do not move freely.
8. Sometimes a tissue fragment remains attached by a few strands of collagen; a second
tungsten needle in a bacteriological loop holder, used freehand, will often detach it.
3.6. Retrieving Dissected Fragments
1. Bring the tip of the pipet close to the fragment to be retrieved and capture the fragment
by suddenly releasing the pipet plunger, dragging fragment and a fixed volume of buffer
into the pipet tip (see Note 9).
2. Expel the captured specimen into a labeled microcentrifuge tube, ready for further
processing (see Note 8).
3. Check that the microdissected specimen is really in the tube. It helps if the fragment
is easily seen with the naked eye. Use a magnifying lens or the microscope to make
certain. Toluidine blue stained fragments are easy to see; unstained fragments may be
practically invisible.
3.7. Extracting DNA: Proteinase K Digestion
In a fixed specimen, nucleic acids are present in a dense array of crosslinked proteins.
Proteinase K digestion appears effectively to release them and make them available
for subsequent PCR.
1. Add an equal volume of Proteinase K digestion buffer pH 8.3 containing 1 mg/mL of
proteinase K and 1 mg/mL Proteinase K to each specimen tube.
2. Digest microdissected specimen in proteinase K (final concentration 500 µg/mL) at 37°C
overnight in a water bath or incubator (digestion can continue over a weekend without
detriment).
3. Heat specimens in a PCR block (95°C for 10), to inactivate PK.
4. Spin the specimen down by brief centrifugation.
5. Specimens are stable at room temperature for subsequent DNA PCR. Store for longer
periods at 4°C or –20°C.
6. Accurate labeling is crucial (see Note 10).
3.8. DNA Purification after PK Digestion
The 25- or 50-µL sample remaining after PK digestion of a microdissected tissue
fragment contains only small quantities of nucleic acids (DNA, RNA). One thousand