34 Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)
453
11. Wash in A. bidest for 2 min at RT.
12. Place the slides in 1% paraformaldehyde for 1 min at 4°C.
13. Wash in A. bidest for 2 min at RT.
14. Dehydrate the slides in a series of 70%, 85% and 100% ethanol at RT,
for 2 min each. Air dry the slides for 10 min at RT.
15. Hybridize with indirectly labeled probes (as described above).
Hybridization
Simultaneous detection of biotin- and digoxigenin-Iabeled probes by Detection
AMCA (blue) and FITC (green):
1. Incubate slides with a mixture of AMCA-conjugated avidin and FITCconjugated monoclonal mouse anti-digoxigenin antibody at RT for
30 min [both 1:200 in PNM buffer].
2. Wash once in PN buffer.
3. Incubate with a mixture of biotinylated goat anti-avidin antibody and
mouse anti-FITC antibody at RT for 30 min [both 1:200 in PNM buffer].
4. Wash once in PN buffer.
5. Incubate with a mixture of AMCA-conjugated avidin and FITC-conjugated donkey anti-mouse antibody at RT for 30 min [both 1:200 in
PNM buffer].
6. Wash once in PN buffer.
Note: The fluorescence intensity can be amplified by multiple repetitions of steps 18 to 20.
7. Mount the slides with antifade solution.
Directly labeled probes can now be counterstained blue by incubating for Counterstaining
5-10 min in DAPI solution (e.g. 6 l DAPI stock solution in 60 ml
2 x SSC) followed by washing for 5-10 min in 2 x sse and mounting in
antifade solution. Alternatively, if green (e.g. FITC) and/or blue (e.g.
AMCA) fluorochromes have been applied, the slides can be mounted
with antifade solution containing 500 ng/ml propidium iodide (red).
If counterstaining is required, the following steps can be included before mounting. Nevertheless, to interfere with the immunophenotype
counterstaining is not generally recommended.