452
BRIGITTE SCHLEGELBERGER et al.
FlalON protocol for combined immunophenotyping and two-color FISH
Indirect immunophenotyping
Monitoring of immunophenotyping
1. Fix slides in fresh acetone for 10 min at room temperature and air dry
for 10 min.
Note: Cryopreserved slides should be air-dried prior to the fIxation for
at least 1 h after being taken out of the freezer.
2. Apply 100 III of monoclonal mouse antibody (against the antigen that is
to be stained) diluted in PNM buffer to the area of the slide containing
the cells or the section. Incubate for 30 min at RT.
3. Wash once in PN buffer.
4. Incubate with Cy3-conjugated goat anti-mouse antibody (1:200 10
PNM buffer) for 30 min.
5. Wash once in PN buffer.
6. Incubate with Cy3-conjugated rabbit anti-goat antibody (1:200 in PNM
buffer) for 30 min.
7. Wash once in PN buffer.
8. Incubate with Cy3-conjugated donkey anti-rabbit antibody (1:100 in
PNM buffer) for 30 min.
9. Wash once in PN buffer.
This four-step immunostaining results in very strong fluorescent staining.
If strongly expressed antigens, such as CD3, have to be stained, it is possible to omit steps 8 and 9 or even steps 6 to 9.
At this point the success of the immunostaining can be monitored. Mount
the slides with PN buffer (not with glycerol!) and examine them under the
fluorescence microscope.
Note: The fluorescence intensity is relatively low if the slides are
mounted in PN buffer. However, mounting in glycerol prior to in
situ hybridization may cause hybridization artifacts.
Monitoring of immunophenotyping is always recommended if the
processed slides have been stored for a long period or if the cells
showed poor morphology prior to the staining procedure.
Hybridization 10. Put the slides into freshly prepared, ice-cold Carnoy's fIxative for
10 min.
BRIGITTE SCHLEGELBERGER et al.
FlalON protocol for combined immunophenotyping and two-color FISH
Indirect immunophenotyping
Monitoring of immunophenotyping
1. Fix slides in fresh acetone for 10 min at room temperature and air dry
for 10 min.
Note: Cryopreserved slides should be air-dried prior to the fIxation for
at least 1 h after being taken out of the freezer.
2. Apply 100 III of monoclonal mouse antibody (against the antigen that is
to be stained) diluted in PNM buffer to the area of the slide containing
the cells or the section. Incubate for 30 min at RT.
3. Wash once in PN buffer.
4. Incubate with Cy3-conjugated goat anti-mouse antibody (1:200 10
PNM buffer) for 30 min.
5. Wash once in PN buffer.
6. Incubate with Cy3-conjugated rabbit anti-goat antibody (1:200 in PNM
buffer) for 30 min.
7. Wash once in PN buffer.
8. Incubate with Cy3-conjugated donkey anti-rabbit antibody (1:100 in
PNM buffer) for 30 min.
9. Wash once in PN buffer.
This four-step immunostaining results in very strong fluorescent staining.
If strongly expressed antigens, such as CD3, have to be stained, it is possible to omit steps 8 and 9 or even steps 6 to 9.
At this point the success of the immunostaining can be monitored. Mount
the slides with PN buffer (not with glycerol!) and examine them under the
fluorescence microscope.
Note: The fluorescence intensity is relatively low if the slides are
mounted in PN buffer. However, mounting in glycerol prior to in
situ hybridization may cause hybridization artifacts.
Monitoring of immunophenotyping is always recommended if the
processed slides have been stored for a long period or if the cells
showed poor morphology prior to the staining procedure.
Hybridization 10. Put the slides into freshly prepared, ice-cold Carnoy's fIxative for
10 min.