33 Simultaneous Multicolor-FISH and Immunocytochemical Analysis of Fresh Tumor Material
435
- Denaturation mixture:
- 70% deionized formamide
- 2xSSC
- 50 mM sodium phosphate buffer pH 7.0
- Store at -20°C
- 65% formamide/2xSSC, pH 7.0
- 50% formamdide/2xSSC, pH 7.0
Procedure
Immunostaining on smear preparations or cryosections
1. Wash the slides in PBS, pH 7.3 for 10 min.
2. Place the slides in a microwave-proof plastic tube containing citrate
buffer solution and treat in a microwave oven for 5 min at 500 W.
3. Reftll with citrate buffer to prevent the probes from drying.
4. Incubate the probes 1 min in the hot buffer.
5. Repeat the microwave treatment under the same conditions.
6. Keep the slides for 20 min in the hot citrate buffer.
7. Rinse the slides for a few seconds with distilled water and then incubate
them in PBS/O.Ol%Triton X-I00 for 10 min.
8. Apply 120 Jll PBS/O.l% BSA to each slide (background blocking) and
incubate 10 min at RT (room temperature) in a moist chamber.
9. Apply the first antibody diluted 1:50-1:100 in PBS/O.l%BSA to the
slides e.g. MIB-l (mouse antibody against Ki-67 antigen; DAKO) or
Do-7 (mouse antibody against p53, DAKO) or mouse anti p16 (Santa
Cruz Biotechnology).
10. Incubate for 1 h at 37°C in a moist chamber (p16 over night at 4°C).
11. Wash briefly 3x5 min with PBS/O.Ol%Triton X-lOO.
12. Apply 120 Jll of the second antibody e.g. goat anti mouse fluorochromel (e.g. AMCA or FITC) diluted 1:50-1:100 in PBS/O.l%BSA
to each slide.
13. Incubate for 1 h at 37°C in a moist chamber.
435
- Denaturation mixture:
- 70% deionized formamide
- 2xSSC
- 50 mM sodium phosphate buffer pH 7.0
- Store at -20°C
- 65% formamide/2xSSC, pH 7.0
- 50% formamdide/2xSSC, pH 7.0
Procedure
Immunostaining on smear preparations or cryosections
1. Wash the slides in PBS, pH 7.3 for 10 min.
2. Place the slides in a microwave-proof plastic tube containing citrate
buffer solution and treat in a microwave oven for 5 min at 500 W.
3. Reftll with citrate buffer to prevent the probes from drying.
4. Incubate the probes 1 min in the hot buffer.
5. Repeat the microwave treatment under the same conditions.
6. Keep the slides for 20 min in the hot citrate buffer.
7. Rinse the slides for a few seconds with distilled water and then incubate
them in PBS/O.Ol%Triton X-I00 for 10 min.
8. Apply 120 Jll PBS/O.l% BSA to each slide (background blocking) and
incubate 10 min at RT (room temperature) in a moist chamber.
9. Apply the first antibody diluted 1:50-1:100 in PBS/O.l%BSA to the
slides e.g. MIB-l (mouse antibody against Ki-67 antigen; DAKO) or
Do-7 (mouse antibody against p53, DAKO) or mouse anti p16 (Santa
Cruz Biotechnology).
10. Incubate for 1 h at 37°C in a moist chamber (p16 over night at 4°C).
11. Wash briefly 3x5 min with PBS/O.Ol%Triton X-lOO.
12. Apply 120 Jll of the second antibody e.g. goat anti mouse fluorochromel (e.g. AMCA or FITC) diluted 1:50-1:100 in PBS/O.l%BSA
to each slide.
13. Incubate for 1 h at 37°C in a moist chamber.