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BRIGITTE SCHLEGELBERGER et al.
1. Wash once in PN buffer and for 5-10 min in 2xSSC.
2. For counterstainingblue incubate the slide for 5-10 min in DAPI solution (6 III DAPI stock solution in 60 ml 2xSSC) (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 nglml propidium iodide (red).
3. Wash for 5-10 min in 2x SSe.
4. Mount the slide with antifade solution.
Note: If necessary, the fluorescence intensity can be amplified by multiple repetitions of steps 3 to 5.
FlalON protocol for combined double-immunophenotyping plus single-color FISH
Immunophenotyping
Immunophenotyping against antigen A by Cy3 (red):
1. Incubate with monoclonal mouse antibody against antigen A (e.g.
CDS) for 30 min at RT.
2. Wash once in PN buffer.
3. Incubate with Cy3-conjugated rabbit anti-mouse antibody (1:200 in
PNM buffer) for 30 min at RT.
4. Wash once in PN buffer.
5. Incubate with Cy3-conjugated donkey anti-rabbit antibody (1:200 in
PNM buffer) for 30 min at RT.
6. Wash once in PN buffer.
7. Incubate with 20% normal mouse serum diluted in PNM buffer for 15
min at RT. By incubating with normal mouse serum containing high
concentrations of mouse immunoglobulins, the free binding sites of
the Cy3-conjugated rabbit anti-mouse antibody (step 2) are blocked.
This prevents the monoclonal mouse antibody against antigen B,
which is applied in step 5, from being caught by free binding sites
of the rabbit anti-mouse antibody.
Note: Attention: Do not wash the slides after this step.
BRIGITTE SCHLEGELBERGER et al.
1. Wash once in PN buffer and for 5-10 min in 2xSSC.
2. For counterstainingblue incubate the slide for 5-10 min in DAPI solution (6 III DAPI stock solution in 60 ml 2xSSC) (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 nglml propidium iodide (red).
3. Wash for 5-10 min in 2x SSe.
4. Mount the slide with antifade solution.
Note: If necessary, the fluorescence intensity can be amplified by multiple repetitions of steps 3 to 5.
FlalON protocol for combined double-immunophenotyping plus single-color FISH
Immunophenotyping
Immunophenotyping against antigen A by Cy3 (red):
1. Incubate with monoclonal mouse antibody against antigen A (e.g.
CDS) for 30 min at RT.
2. Wash once in PN buffer.
3. Incubate with Cy3-conjugated rabbit anti-mouse antibody (1:200 in
PNM buffer) for 30 min at RT.
4. Wash once in PN buffer.
5. Incubate with Cy3-conjugated donkey anti-rabbit antibody (1:200 in
PNM buffer) for 30 min at RT.
6. Wash once in PN buffer.
7. Incubate with 20% normal mouse serum diluted in PNM buffer for 15
min at RT. By incubating with normal mouse serum containing high
concentrations of mouse immunoglobulins, the free binding sites of
the Cy3-conjugated rabbit anti-mouse antibody (step 2) are blocked.
This prevents the monoclonal mouse antibody against antigen B,
which is applied in step 5, from being caught by free binding sites
of the rabbit anti-mouse antibody.
Note: Attention: Do not wash the slides after this step.