33 Simultaneous Multicolor-FISH and Immunocytochemical Analysis of Fresh Tumor Material
439
7. Incubate for 1 h at 37°C in a moist chamber.
8. Wash 3x5 min with 4xSSC/0.050/0 Tween-20.
9. Apply 120 III streptavidin-fluorochrome 2 (e.g. FITC or AMCA) diluted
1:100 and anti-digoxigenin-fluorochrome 3 (e.g. rhodamine) diluted
1:25 in 4xSSC/0.50/0 blocking reagent to each slide.
10. Incubate for 1 h at 37°C in a moist chamber.
11. Wash briefly 3x5 min with 4xSSC/0.050/0 Tween-20.
12. Dehydrate the slides in an ethanol series and air dry.
13. Apply 25 III embedding medium (e.g. Vectashield, Vector) and cover
with a 24 x 60 coverslip.
Troubleshooting
Immunostaining
If no or weak signals are observed, then:
- Raise the concentration of the primary antibody.
- Raise the incubation time of the primary antibody (up to 16 h at 37°C,
RT or 4°C).
- Modify the incubation time ofthe postfIxation step after immunostainmg.
Multicolor-FISH
If no signals are observed, then:
- Amplify the signals in an additional step.
- Check the labeling quality of the assigned in situ probes (dot blot).
- Dot blot protocol:
- 1. Spot serial dilution e.g. 6 ng, 0.6 ng, 60 pg, 6 pg, 0.6 pg, and 0.06 pg
of the labeled probe on a nitrocellulose or nylon membrane.
- 2. Fix the probes to the membrane by baking for 60 min at BO°C on a
heating plate or by cross-linking with UV light.
- 3. Incubate the membrane in 6xSSC/lO/OBSA for 30 min.