Biology Reference
In-Depth Information
2. Materials
General Materials
1. Glass slides, glass coverslip (22-mm square), and standard implements for
preparing embryos for agar mounts (see the first section of this chapter).
2. High-pressure freezing specimen carriers.
3. l-hexadecene. l-hexadecene is used to coat the top specimen carrier to promote
release of carriers after freezing and as filler in the bottom carrier as described
by
McDonald (1999)
4. High-pressure freezer (we used the BAL-TEC HPM 010).
5. Petri dishes to make a humid chamber (95 and 60-mm diam. dishes).
6. 20-mL scintillation vials for resin infiltration.
7. 3-Aminopropyltriethoxy-silane, 3-APTS (Sigma, A-3648).
8. Agarose (1% gel strength of 1000 g/cm
2
or greater for base; Invitrogen, cal. no.
15510-027). The strength of agarose can be tested empirically by making a thin
pad of agarose over a slide. If the entire pad can be lifted off the slide without
tearing, using a razor blade under one corner of the pad, it is strong enough to be
used as a base for the correlative pad. We start with a 5% solution of agarose and
dilute to between 4% and 5% with additional 0.1M HEPES as needed to form
the base pad.
9. Low melting temperature agarose (Sigma, A-9539) to immobilize embryos.
10. 2 mL polypropylene vials with screw cap lids for freeze substitution.
11. Styrofoam box, dry ice, rotary shaker in 4
C cold room. Freeze substitution
using a Styrofoam box has been described by
McDonald (1999)
. We fit the
aluminum block into a tight-fitting piece of Styrofoam, which holds the block in
place as the dry ice sublimes. We do not monitor the temperature during freeze
substitution.
12. Disposable polyethylene pipettes (Fisher, cat no. 12-711-7). Any 3 mL or
smaller disposable pipette with its own bulb should suffice. The use of
disposable pipettes for dispensing fixatives avoids contaminating more
expensive pipettes.
13. Microtiter plate shaker and smaller Styrofoam box in
20
C freezer.
14. Single-edged razor blade. A single-edged razor blade is used to cut out a small
piece of agarose containing the embryos to fit within the 2 mm specimen holder.
The edge of a razor blade or a fine-tipped weighing spatula can also be used to
transfer to the specimen carrier.
15. Sharpened tooth picks.
16. Rain-X (UnelkoCorp., Scottsdale, AZ) to coat slides for flat embedding. Two
standard 1
3-inch microscope slides are coated with Rain-X or Teflon release
agent using a cotton tipped applicator or Kimwipe. Coat each slide 3
and buff
clear with a Kimwipe. This coating prevents the epoxy resin from gluing the
slides together.
17. Clear acetate tape (Scotch brand or similar) for use as a spacer.
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