440
HElKE STEILEN-GIMBEL and STEFFI URBSCHAT
- 4. Incubate the membrane in buffer 1 (0.05 M Tris, 0.075 M NaCl,
pH 7.5) for 10 min
- 5. Dilute Streptavidin-alkaline phosphatase (2.25 U) or Anti-DIGalkaline phosphatase (2.25 U) in 3 ml buffer 1.
- 6. Incubate the membrane in diluted antibody solution for 30 min.
- 7. Wash 2x5 min in buffer 1.
- 8. Wash 1x 5 min in buffer 2 (0.05 M Tris, 0.05 M NaCI, 0.025 M
MgCh, pH 9.5).
- 9. Mix 33 ,. .tl NBT (25 mg/ml, Roche Molecular Biochemicals) and
25 J.lI BCIP (50 mg/ml, Roche Molecular Biochemicals) with 3 ml
buffer 2 (prepare this solution freshly!).
- 10. Incubate membrane in stain solution for 30-60 min.
- 11. Fix membrane in 70% ethanol.
- Check the fragment length of the labeled probe in agarose gel.
- Modify the denaturation step of the immunostained slides by varying
temperature and denaturation time of the procedure.
If you have a high background fluorescence, then:
- Spin down the diluted fluorochrome-conjugated antibody (2 min,
14.000 rpm) and take only the supernatant (this avoids background
induced by agglutinated fluorochromes).
- Reduce the concentration of primary and fluorochrome-conjugated
antibodies.
- Make the wash steps between signal detection longer and at a higher
temperature, e.g., 3 xl0 min at 37°C.
- Store the mounted slides in a dark moist chamber at 4°C for a few days
and observe under the microscope again.
Acknowledgements. We thank K.-D. Zang and W. Henn for helpful discussions, and B.
Mayer and B. Rohrschneider for technical assistance.
References
Balazs M, Mayall BH, Waldman FM (1991) Simultaneous analysis of chromosomal
aneusomy and 5-bromodeoxyuridine incorporation in MCF-7 breast tumor cell
line. Cancer Genet Cytogenet 57:93-102
Dirks RW, Van de Rijke FM, Fujishita S, Van der Ploeg M, Raap AK (1993) Methodologies for specific intron and exon RNA localization in cultured cells by haptenized
and fluorochromized probes. J Cell Sci 104:1187-97
HElKE STEILEN-GIMBEL and STEFFI URBSCHAT
- 4. Incubate the membrane in buffer 1 (0.05 M Tris, 0.075 M NaCl,
pH 7.5) for 10 min
- 5. Dilute Streptavidin-alkaline phosphatase (2.25 U) or Anti-DIGalkaline phosphatase (2.25 U) in 3 ml buffer 1.
- 6. Incubate the membrane in diluted antibody solution for 30 min.
- 7. Wash 2x5 min in buffer 1.
- 8. Wash 1x 5 min in buffer 2 (0.05 M Tris, 0.05 M NaCI, 0.025 M
MgCh, pH 9.5).
- 9. Mix 33 ,. .tl NBT (25 mg/ml, Roche Molecular Biochemicals) and
25 J.lI BCIP (50 mg/ml, Roche Molecular Biochemicals) with 3 ml
buffer 2 (prepare this solution freshly!).
- 10. Incubate membrane in stain solution for 30-60 min.
- 11. Fix membrane in 70% ethanol.
- Check the fragment length of the labeled probe in agarose gel.
- Modify the denaturation step of the immunostained slides by varying
temperature and denaturation time of the procedure.
If you have a high background fluorescence, then:
- Spin down the diluted fluorochrome-conjugated antibody (2 min,
14.000 rpm) and take only the supernatant (this avoids background
induced by agglutinated fluorochromes).
- Reduce the concentration of primary and fluorochrome-conjugated
antibodies.
- Make the wash steps between signal detection longer and at a higher
temperature, e.g., 3 xl0 min at 37°C.
- Store the mounted slides in a dark moist chamber at 4°C for a few days
and observe under the microscope again.
Acknowledgements. We thank K.-D. Zang and W. Henn for helpful discussions, and B.
Mayer and B. Rohrschneider for technical assistance.
References
Balazs M, Mayall BH, Waldman FM (1991) Simultaneous analysis of chromosomal
aneusomy and 5-bromodeoxyuridine incorporation in MCF-7 breast tumor cell
line. Cancer Genet Cytogenet 57:93-102
Dirks RW, Van de Rijke FM, Fujishita S, Van der Ploeg M, Raap AK (1993) Methodologies for specific intron and exon RNA localization in cultured cells by haptenized
and fluorochromized probes. J Cell Sci 104:1187-97