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

36 Going
3. Methods
3.1. Section Cutting
Careful clean techniques should be used when cutting sections for PCR analysis.
1. Use a new part of the microtome blade to cut each section to avoid possible the carryover
of DNA from one tissue block to sections of the next.
2. Ribbons of sections should be floated out on a clean water bath and no buildup of section
debris permitted.
3. Glass slides upon which sections are mounted should be scrupulously clean. Dry mounted
sections at 56°C for 2 h (see Note 1).
3.2. Dewaxing and Staining Sections
Dewax sections completely before microdissection.
1. Immerse 6- to 7-µm sections in a slide rack for 10 min in xylene. Drain surplus xylene
thoroughly from the sections to minimize the carryover of dissolved wax and then transfer
to a second xylene bath for 2 min. Avoid breathing xylene vapor and be aware of the fire
hazards of organic solvents.
2. Take sections through two baths of 99% industrial methylated spirits to remove xylene
and a third bath of 95% industrial methylated spirits.
3. Transfer sections to distilled or deionized water of a satisfactory standard for preparing
PCR reagents. Stain by immersion for 30 s in 0.05% w/v toluidine blue in distilled water
(see Note 2).
4. Wash in distilled water (see Note 3).
5. After dissection, dehydrate and mount slides with a coverslip to provide a permanent
record of the dissection (see Note 4).
3.3. Microdissection Tools
Successful dissection can be conducted freehand using sterile curved scalpel blades
or sterile hypodermic needles, with or without a dissecting microscope.
1. Take a section for dissection from the water bath immediately before it is needed, drain
it, and blot any surplus water from around the section with a disposable, lint-free tissue
(do not touch the section).
2. Stroke the edge of the scalpel blade decisively across the section to remove a strip of tissue,
the width of which will vary with the angle between blade and section (see Note 5).
3. For more precise dissection with the micromanipulator, an electrolytically sharpened
tungsten needle is ideal (Fig. 1). This tool consists of 25 mm of tungsten wire, 0.5 mm
in diameter, sharpened and polished electrolytically to a fine point (tip radius several
microns).
4. Mount the needle in a collet-type bacteriological loop holder.
5. Mount the loop holder in the tool holder of the micromanipulator and angle it downward
25 to 30º.
3.4. Making and Maintaining Tungsten Microneedles
Tungsten needles can be obtained ready-made as ohmic probe needles but are easily
fabricated from plain 0.5-mm tungsten wire (obtainable from suppliers of equipment
for electron microscopy) This requires an electrolytic cell (see Note 6).