InSitu Protocol

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 minutes. 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 jars. Put on at least 5uL per section and coverslip. Put slides in moist-chamber and incubate at 37 degrees C for 60 minutes. 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 70% ethanol/ .3M ammonium acetate 95% ethanol/ .3M ammonium acetate 100% ethanol Let slides dry on bench for at least an hour. 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.

Emulsion Dipping:

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.

Developing Slides:

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 dip Dish 3-> Rapid Fix 2minutes Dish 4-> dH20 dip Scrape emulsion off back of slide and front edge using razor blade. Stain lightly in .5% Toluidine blue. Rinse in dH20. 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 remaining slides.