34 Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)
451
2. Wash once with PN buffer.
3. Incubate with a mixture of biotinylated goat anti-avidin antibody
(1:200 in PNM buffer) and digoxigenin-conjugated sheep-anti mouse
antibody at RT for 30 min (1:100 in PNM buffer).
4. Wash once with PN buffer.
5. Incubate slides with a mixture of Cy3-conjugated avidin (1:400 in PNM
buffer) and FITC-conjugated sheep anti-digoxigenin antibody (1:100
in PNM buffer) at RT for 30 min.
6. Wash once in PN buffer and for 5-10 min in 2xSSC.
7. For counterstaining blue incubate the slide for 5-10 min in DAPI-Solution (6111 DAPI stock solution in 60 mI2xSSC) (DAPI-concentration
and/or incubation time can be adjusted according to preferred staining
intensity).
For counterstaining red - if the hybridization signals are visualized by
AMCA and FITC- mount the slides with antifade solution containing
500 ng/ml propidium iodide (red).
8. Wash for 5-10 min in 2xSSC.
9. Mount the slide with antifade solution.
Note: If necessary, the fluorescence intensity can be amplified by multiple repetitions of steps 3 to 5.
Slide preparation for FlalON
The quality of slides is even more important for successfully performing
the FICTION technique than for FISH. For FICTION, cytospin slides,
smears, imprints and cryostat sections may be used. Freshly prepared
slides should be air-dried overnight at room temperature. Thereafter,
slides can be processed immediately or stored at -80
a C. Slides preserved
in this way can be used for years; when single slides are taken out of the
freezer, it is very important to make sure that the remaining slides in the
freezer do not thaw. This is possible by taking the slides out of the container inside the freezer. If slides are stored at -20
D C, the quality of the immunophenotyping may be diminished after a few months. However,
the quality of the hybridization is not affected. Before starting the FICTION procedure, slides should be examined by phase contrast microscopy
for the morphological quality of the cells. If many slides are available, only
those with good cell morphology should be processed.
451
2. Wash once with PN buffer.
3. Incubate with a mixture of biotinylated goat anti-avidin antibody
(1:200 in PNM buffer) and digoxigenin-conjugated sheep-anti mouse
antibody at RT for 30 min (1:100 in PNM buffer).
4. Wash once with PN buffer.
5. Incubate slides with a mixture of Cy3-conjugated avidin (1:400 in PNM
buffer) and FITC-conjugated sheep anti-digoxigenin antibody (1:100
in PNM buffer) at RT for 30 min.
6. Wash once in PN buffer and for 5-10 min in 2xSSC.
7. For counterstaining blue incubate the slide for 5-10 min in DAPI-Solution (6111 DAPI stock solution in 60 mI2xSSC) (DAPI-concentration
and/or incubation time can be adjusted according to preferred staining
intensity).
For counterstaining red - if the hybridization signals are visualized by
AMCA and FITC- mount the slides with antifade solution containing
500 ng/ml propidium iodide (red).
8. Wash for 5-10 min in 2xSSC.
9. Mount the slide with antifade solution.
Note: If necessary, the fluorescence intensity can be amplified by multiple repetitions of steps 3 to 5.
Slide preparation for FlalON
The quality of slides is even more important for successfully performing
the FICTION technique than for FISH. For FICTION, cytospin slides,
smears, imprints and cryostat sections may be used. Freshly prepared
slides should be air-dried overnight at room temperature. Thereafter,
slides can be processed immediately or stored at -80
a C. Slides preserved
in this way can be used for years; when single slides are taken out of the
freezer, it is very important to make sure that the remaining slides in the
freezer do not thaw. This is possible by taking the slides out of the container inside the freezer. If slides are stored at -20
D C, the quality of the immunophenotyping may be diminished after a few months. However,
the quality of the hybridization is not affected. Before starting the FICTION procedure, slides should be examined by phase contrast microscopy
for the morphological quality of the cells. If many slides are available, only
those with good cell morphology should be processed.