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Basic Protocol 5:
DETECTION OF RADIOLABELED PROBES
Detection of radiolabeled probes entails apposition of the samples to x-ray film or phosphorimaging plates and subsequent development. Higher, cellular resolution entails coating of the sample with a nuclear emulsion (described here). These steps are performed after detection of the digoxigenin-labeled probes if the two types of probes are used simultaneously.
Materials
Prepare the nuclear emulsion for coating the sections
1. Under safelight conditions, scoop out 40 ml of emulsion with spatula into a coplin jar containing 1.6 ml of 7.5M ammonium acetate (final concentration of 300mM).
2. Place the coplin in a 40° C water bath for 20-30 mins to allow air bubbles to rise. Mix gently and look for bubbles on a clean slide after dipping it into the emulsion.
3. Dip slides into the emulsion, and stand up for several hours to dry.
We place 5 slides in red plastic slide grips and dip them 5 at a time into the emulsion. We then hang them from a custom-made plexiglass holder.
4. The emulsion-coated slides are placed in black slide boxes with desiccant capsules. Tape the edges of the box with black photography tape and store the boxes at 4° C in the dark.
Develop the emulsions and stain the tissue sections
5. Put the slides in racks and pass through the solutions as follows at 17° C. (with agitation every 30s): D-19 for 2mins; running tap water 15s with slight agitation; and Kodak Rapid Fix (without hardener) for 2mins.
The room lights may be turned on after all slides are fixed.
6. Rinse in running tap water for 8 minutes. Counterstain, if desired, for 30s in 0.4% toluidine blue, 2µg/ml ethidium bromide, hematoxylin/eosin, or stain of choice, and rinse again briefly to remove excess stain.
Some stains may obscure or destroy colorimetric detection of the digoxigenin probe or silver grains (e.g., periodic acid Schiff, cresyl violet).
7. Dip very briefly into deionized water, then 70% ethanol and place on slide warmer to thoroughly dry.
8. Coverslip slides with Cytoseal 60 (or, similar organic-based) mountant.
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REAGENT AND SOLUTIONS
Use DEPC-treated H2O to make solutions for solutions for pretreatment and hybridization. De-ionized H2O may be used for the subsequent wash and digoxigenin development steps.
Slides for mounting tissue sections:
- Subbed slides
- Place slides in racks and soak in soap solution for 1 hr.
- Rinse in deionized water. Change the water several times to be sure that all of the soap is removed.
- Dissolve 1.88 g of gelatin (300 bloom swine) in 750 ml hot H2O (do not allow to boil). Cool and dissolve 0.188 g CrK(SO4)2*12H2O in the solution.
- Dip the slides into the subbing solution, drain the slides onto a paper towel and allow to air dry for 1 hr.
- Dip the slides into the subbing solution again. Drain and cover loosely with plastic wrap or bench paper.
- When thoroughly dry, store the slides in slide boxes.
- Silanized slides:
- Clean slides with a lint free cloth manually (lots of work, but needed) and put them in racks.
- Dip slides in 2% aminoalkalynsilane (Sigma A-3648) in dry acetone for 10 sec.
- Rinse in deionized water 3 times.
- Air dry overnight and store in boxes protected from dust.
- Positively charged slides:
- We buy these slides: Superfrost Plus microscope slides (4951 , Erie Scientific, Portsmouth, NH 03801) or you can make them as follows:
- Clean slides with a lint free cloth manually (lots of work, but needed) and put them in racks
Dip slides in 50?g/ml poly-L-lysine
- Air dry overnight and store in boxes protected from dust.
10X Phosphate-buffered saline, pH 7.4 (PBS)
