Cut 5 micron thick sections
and freeze at –80
degrees C overnight. Use gelatin-coated slides, and
be sure to cut duplicates and place section at far-side of
slide away from label.
Incubate in 2% paraformaldehyde from an 8% stock for 10
Make a 1:30,000 dilution of the DNAse with reactin buffer
(Sigma D7291) at 25 deg C.
Treat with DNAse solution for five minutes.
Wash once in 1x PBS containing 2mg/mL glycine using coplin
Put on at least 5uL per section and coverslip. Put slides
in moist-chamber and incubate at 37 degrees C for
At 37 deg C, wash slides for 2 hours with 4-5 changes of
prewarmed DNA wash solution.
Do a dehydration series with ethanol for
two minutes each as follows:
50% ethanol/ .3M ammonium acetate
ethanol/ .3M ammonium acetate
95% ethanol/ .3M
Let slides dry on bench for at least
Tape slides face down onto film and
expose overnight in gel-cartridge.
Develop film. If tissue section is visible
on film and looks ok, incubate in emulsion for four days.
If tissue section either not visible or too dark on film,
readjust emulsion incubation time as appropriate.
Bring water bath to 45 degrees C in darkroom. While water
bath is heating, remove emulsion aliquot from refrigerator
and allow to come to room temperature (takes about 40 minutes).
When water bath and emulsion reach temperature, make a 1:1
dilution of the emulsion with dH20. Invert once carefully. Be
careful not to agitate emulsion too much as this will increase
background and cause bubbles to form! Transfer emulsion
aliquot to water bath and allow it to reach 45 degrees
C (about 20 minutes).
Before dipping slides, test emulsion for bubbles
by dipping a blank slide. If slide has bubbles on it,
wait another ten minutes and dip another blank slide. Continue
until no or very few bubbles are present.
Dip relevant slides
as well as 2-3 blank slides in emulsion. Allow to
dry for 1-4 hrs. Check for dryness by checking blank
slides. When dry, place in light-tight box with dessicant “cigarette” and
store at 4 degrees C for appropriate amount of
time based on film reading. Be sure to store slides in a refrigerator
not containing radioactive material, and be sure not to agitate
slides as the emulsion is sensitive to shock.
Develop only one of the duplicate test slides
that should be positive to be sure slides are ready for development.
There should not be an excess or an absence of labeling.
In darkroom, allow boxes to come
to room temperature in boxes to avoid condensation which
causes image fading.
Using D-19 stored at 4 deg C, dilute 1:1
and filter. Cool D-19 to 16 deg C. Setup four staining dishes
and dip as follows:
Dish 1-> D-19 3.5 minutes
Dish 2-> dH20
Dish 3-> Rapid Fix 2minutes
Dish 4-> dH20
Scrape emulsion off back of slide and
front edge using razor blade.
Stain lightly in .5% Toluidine blue. Rinse
Dehydrate with ethanol as follows: 30,
50, 70, 85, 95, 100, 100
Do three final Xylene steps.
Coverslip with cytoseal.
If labeling is sufficient, repeat with