456
BRIGITTE SCHLEGELBERGER et al.
22. Wash in PN buffer.
23. Incubate with FITC-conjugated donkey anti-mouse antibody at RT for
30 min (1:200 in PNM buffer).
24. Wash in PN buffer.
25. Incubate with monoclonal mouse anti-FITC antibody for 30 min at RT
(1:200 in PNM buffer).
26. Wash in PN buffer.
27. Incubate with FITC-conjugated donkey anti-mouse antibody for 30
min at RT (1:200 in PNM buffer).
28. Wash in PN buffer.
Note: If required, the fluorescence intensity can be amplified by repeating steps 25 to 28.
29. Mount the slides with antifade solution.
Storage
of stained slides
Mounted slides can be stored for several weeks at 4
D C and for several
months or up to years at -20
D
e. Always keep slides in the dark.
Other FICTION protocols
The FICTION technique can generally be performed with any kind ofFISH
procedure (Fig. 1). If immunophenotyping and, most important, paraformaldehyde fixation after immunophenotyping is done according to
the FICTION protocol, then it is possible to proceed with individual
FISH protocols as established in other laboratories. This way, all types
of probes for detecting numerical or structural aberrations can be employed. The only restriction is that proteolytic treatment must be avoided
because it would considerably impair the quality of immunophenotyping.
Results
Evaluation of FICTION analyses
Technical prerequisites: In general, the different fluorescent colors of the
slides can be evaluated separately under each fluorescence microscope
equipped with specific ftlter sets for AMCA, FITC and Cy3. Alternatively,
if triple-dye fluorescence filter sets are used, all three fluorescent colors