34 Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)
455
Immunophenotyping against antigen B by AMCA (blue):
8. Incubate with biotinylated monoclonal antibody against antigen B (e.g.
CD19) in PNM buffer containing 20% normal mouse serum for 30 min
at RT.
9. Wash in PN buffer.
Note: The detection system is very similar to that used for the detection
of biotinylated DNA probes.
10. Incubate with AMCA-conjugated avidin (1:200 in PNM buffer) for 30
min at RT.
11. Wash in PN buffer.
12. Incubate with biotinylated goat anti-avidin antibody (1:200 in PNM
buffer) for 30 min at RT.
13. Wash in PN buffer.
Note: Multiple amplifications of the AMCA staining are possible by
repeating steps 10 to 13.
14. Fix in 1% paraformaldehyde for 1 min at 4°C.
15. Wash in A. bidest for 2 min at RT.
16. Put the slides into fresh ice-cold Carnoy's fixative for 10 min.
17. Wash in A. bidest for 2 min at RT.
18. Dehydrate the slides in a series of 70%, 85% and 100% ethanol at RT for
2 min each.
19. Air dry the slides for 10 min at RT.
20. Apply hybridization mixture containing digoxigenin labeled probe to
the slide.
Note: Attention: In this protocol a digoxigenin labeled probe is applied. Do not use biotin-labeled probes, because they bind nonspecifically to the antibody cascade of antigen B.
Detection of the digoxigenin labeled probe by FITC (green):
21. Incubate with an FITC-conjugated monoclonal mouse anti-digoxigenin antibody at RT for 30 min (1:200 in PN buffer).
Hybridization
Detection
455
Immunophenotyping against antigen B by AMCA (blue):
8. Incubate with biotinylated monoclonal antibody against antigen B (e.g.
CD19) in PNM buffer containing 20% normal mouse serum for 30 min
at RT.
9. Wash in PN buffer.
Note: The detection system is very similar to that used for the detection
of biotinylated DNA probes.
10. Incubate with AMCA-conjugated avidin (1:200 in PNM buffer) for 30
min at RT.
11. Wash in PN buffer.
12. Incubate with biotinylated goat anti-avidin antibody (1:200 in PNM
buffer) for 30 min at RT.
13. Wash in PN buffer.
Note: Multiple amplifications of the AMCA staining are possible by
repeating steps 10 to 13.
14. Fix in 1% paraformaldehyde for 1 min at 4°C.
15. Wash in A. bidest for 2 min at RT.
16. Put the slides into fresh ice-cold Carnoy's fixative for 10 min.
17. Wash in A. bidest for 2 min at RT.
18. Dehydrate the slides in a series of 70%, 85% and 100% ethanol at RT for
2 min each.
19. Air dry the slides for 10 min at RT.
20. Apply hybridization mixture containing digoxigenin labeled probe to
the slide.
Note: Attention: In this protocol a digoxigenin labeled probe is applied. Do not use biotin-labeled probes, because they bind nonspecifically to the antibody cascade of antigen B.
Detection of the digoxigenin labeled probe by FITC (green):
21. Incubate with an FITC-conjugated monoclonal mouse anti-digoxigenin antibody at RT for 30 min (1:200 in PN buffer).
Hybridization
Detection