11 Formalin-Fixed and Paraffin-Embedded Tissue Sections
153
11. Add lyophilized pepsin to the bottle of prewarmed 0.9% NaCI, pH 2.0, Protease treatment
to a final concentration of about 1250-1750 units/ml, invert several
times to mix and pour into the prewarmed Coplin jar.
12. Remove slides from 2x SSC, blot off excess liquid and incubate in pepsin solution at 37°C: 10 min.
Note: Make sure that temperature has reached 37°C after adding all
slides (at most 6/jar) and check temperature during the whole procedure.
13. Rinse in 2x SSC at room temperature: 2x 5 min.
14. Place on the slide warmer to dry: 2 min.
15. Immerse slides in 3.7% buffered formalin solution at room tempera- Postfixation
ture: 10 min.
Note: Do not shake Coplin jars during this step.
16. Rinse in 2x SSC at room temperature: 2 x 5 min.
17. Place on the heating plate to dry: 2 min.
18. Place slides into 70% formamidel2 x SSC at 73°C: 5 min.
Note: Make sure that temperature has reached 7l-73°C after adding all
slides (at most 6/jar) and check temperature during the whole procedure.
19. Dehydrate slides in icecold 70%, 85% and 100% ethanol: 1-2 min each.
20. Dry slides at room temperature.
Note: Ifworking with direct fluorochrome labeled probes, all following
steps should be performed protected from the light (in subdued light
or in a dark room if possible).
Denaturation
21. Prepare 10-15 Jll hybridization mixture containing labeled probe, Hybridization
blocking DNA, 50% formamide and 10% dextran sulfate. If commercially available probes are used, prepare according to the manufacturers recommendations (e.g. in the cases demonstrated in Figs.
3A,B a mixture of centromere and locus specific probes was used.
22. Cap tightly and denature at 73°C in a water bath: 5 min.
23. Apply denaturated hybridization mixture onto the slide.
153
11. Add lyophilized pepsin to the bottle of prewarmed 0.9% NaCI, pH 2.0, Protease treatment
to a final concentration of about 1250-1750 units/ml, invert several
times to mix and pour into the prewarmed Coplin jar.
12. Remove slides from 2x SSC, blot off excess liquid and incubate in pepsin solution at 37°C: 10 min.
Note: Make sure that temperature has reached 37°C after adding all
slides (at most 6/jar) and check temperature during the whole procedure.
13. Rinse in 2x SSC at room temperature: 2x 5 min.
14. Place on the slide warmer to dry: 2 min.
15. Immerse slides in 3.7% buffered formalin solution at room tempera- Postfixation
ture: 10 min.
Note: Do not shake Coplin jars during this step.
16. Rinse in 2x SSC at room temperature: 2 x 5 min.
17. Place on the heating plate to dry: 2 min.
18. Place slides into 70% formamidel2 x SSC at 73°C: 5 min.
Note: Make sure that temperature has reached 7l-73°C after adding all
slides (at most 6/jar) and check temperature during the whole procedure.
19. Dehydrate slides in icecold 70%, 85% and 100% ethanol: 1-2 min each.
20. Dry slides at room temperature.
Note: Ifworking with direct fluorochrome labeled probes, all following
steps should be performed protected from the light (in subdued light
or in a dark room if possible).
Denaturation
21. Prepare 10-15 Jll hybridization mixture containing labeled probe, Hybridization
blocking DNA, 50% formamide and 10% dextran sulfate. If commercially available probes are used, prepare according to the manufacturers recommendations (e.g. in the cases demonstrated in Figs.
3A,B a mixture of centromere and locus specific probes was used.
22. Cap tightly and denature at 73°C in a water bath: 5 min.
23. Apply denaturated hybridization mixture onto the slide.