7 In Situ Hybridization for RNA: Radioactive DNA Probe
107
Cover glass for in situ hybridization
1. Wipe off cover glass with clean gauze
2. Put in fresh (or newly made) 100% ethanol overnight
3. Dry in air by placing them on a clean filter or paper towel
4. Dip cover slides into Sigma coat ( kept at 4°C)
5. Dry in air
6. Heat treatment: This heat treatment should be performed
prior to actual ISH
Procedure of in situ hybridization
1. Cut the paraffin block to 4 mm thickness
2. Mount on subbed slides
3. Dry slides thoroughly for 3 hrs on a slide warmer at 42°C
4. Store at 4°C until use
1. Deparaffinize by placing slides in a Caplin jar and heating in
an oven at 60°C for 1 hr
2. Add fresh xylene to the hot Caplin jar and leave the jar under a
ventilation hood for 30 mins with occasional shaking (for removing melted paraffin)
3. Wash in fresh xylene (15 mins x2) and in 100% ethanol
(5 mins x2)
4. Dry in air for 10 mins
5. Fix in 4% paraformaldehyde in PBS for 10 mins (attach the
sections to slides firmly)
6. Wash in PBS (5 mins x3)
7. Dehydrate in 70% ethanol (5 mins x2) and in 95% ethanol
(5 mins)
8. Dry in air
Sectioning
In situ hybridization -
paraffin
section