Section on Neural Gene Expression
Autoradiography using Emulsion-coated Coverslips
1. Cleaning coverslips (use gloves)
- Add 1 bottle of Chromerge, 1/5 at a time, into 2.5 L
bottle of Sulfuric Acid. Invert the bottle to mix
after each addition.
- Load coverslips into glass carriers in every other slot
at a slant (see fig. 1).
- Pour enough chromic acid solution into tray to cover
the tops of the coverslips. Leave overnight.
- Take carriers out of acid and place into a tray containing
about one inch of water. Do not touch the coverslips!
- Rinse in running tap water 20 minutes, empty water
and rinse again for 3 hours. Wash with 3 changes of
distilled or deionized water. Place tray on a slant
(to allow water to drain) into 60° C oven overnight.
- Once the coverslips have been acid cleaned it is essential
that the end which will be dipped in emulsion not be
touched, and that it be as clean as possible (i.e.,
not the end which was in the corner of the slide carrier
(see fig. 2).
- After drying, place coverslips into black slide boxes.
Touch only one end of each, and place the touched end
toward the left side of the slide box.
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Figure 1
Figure 2
Touch the slips only at the end marked*
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2.Dipping (in a dark room)
- Turn on water bath to 42° C.
- Melt NTB-2 or NTB-3 emulsion
(which had been diluted 1:1 with H2O) by placing covered
50 ml plastic Falcon tube of emulsion in a beaker of
water in the water bath for about 20 minutes. Gently
invert the tube of emulsion several times to mix, then
slowly pour the emulsion into a coplin jar. Let the
emulsion stand 20-30 minutes before use to allow air
bubbles to rise. The emulsion may be mixed further
with a clean slide.
- Be sure to touch only the left side of each coverslip.
Dip one coverslip and examine for air bubbles. If
there are still bubbles, wait a while, then test again.
If there are no bubbles, you are ready to begin.
- Dip the coverslips (or slides) slowly to prevent forming
air bubbles. Lower the coverslip until it touches
the bottom of the jar. Raise slowly, and drain off
the excess onto the lipof the jar (see fig. 3).
- Hang the dipped coverslips on the clothespin rack to
dry for 3 hours. Place 10-15 dried coverslips into
each black box with Drierite capsules. Tape edges of
the box with black photography tape. Store boxes at
4° C in a light safe.
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Figure 3
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3.Coverslipping slides (in a dark room)
- With the light on, draw a heavy line with a wax pencil
across each slide to prevent the super glue from spreading
past the end of the slide. Lay all slides face up
on a reflective metalsheet or mirror. Place a drop
of super glue onto the frosted end of each slide (see
fig.4).
- In the dark, open the box of coverslips and, holding
the coverslips only at the undipped end, carefully
place one coverslip on each slide. The emulsion coated
end of the coverslip should extend about 2 mm over
the end of the slide.
- Place two slides back to back, place one glass square
on the coverslip over the tissue on each side, and clip
the assembly together (see fig.5). Place these assemblies
in a slide box with Drierite capsules, and tape the
edges of the box with opaque photography tape. Store
boxes at 4° C in a light safe.
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Figure 4
Figure 5
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4. Developing (in a dark room)
- Dilute the full strength Dektol developer 1:1 with water.
Use KODAK Fixer full strength.
- Cool one dish of
each of the above solutions plus one dish of water
to 17° C.
In the dark:
- Unclip paired slides and remove
glass squares. Carefully separate the free edge of
the coverslip from the slide with a razor blade. Insert
a short piece of toothpick between the slide and coverslip
to prop open (see fig. 6)
- Put these slides back to back in pairs in the slide
holder.(see fig. 7)
- Put the slides through the solutions as follows: Dektol,
2 minutes; water, 5 seconds with slight agitation;
Kodak Fixer, 3 minutes.
- (You may turn on the lights after all slides are fixed)
- Rinse in running tap water for 18 minutes. Counterstain,
if desired, for 30 seconds in toluidin blue or stain
of choice, and rinse again briefly to remove excess
stain. Blot off excess water, and place slides on
a slide warmer to dry. Dip in xylene and coverslip
dipped slides or reappose emulsion-coated coverslips
with mountant.
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Figure 6
Figure 7
coverslipped slides, back to back
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REFERENCES
1. Young, W.S.,III and Kuhar, M.J. A new method for
receptor autoradiography: [3H]-opioid receptors in
rat brain. Brain Res. 179:255-270, 1979.
Materials
- 25 x 75 mm coverglass, thickness #1 Freed (Diamond
Brand, PGC Scientifics, 301-840-1111)
- Chromerge 1 oz. bottle (11-4750-01 Pkg. 6 bottles,
Ace Scientific) to 9 lb container concentrated sulfuric
acid.
- Humi-caps (245-1, United Dessicants 609-662-6500)
- Ilford emulsions (Polysciences, 1-800-523-2575)
