8 Amniotic Fluid Cells - Uncultured
123
6. Tip off the stain solution into a bottle for special waste disposal (DAPI
is a possible mutagen) and place the slides into a Coplin jar. Rinse 3
times with deionized water.
7. Let the slides dry in the dark.
8. Apply 20 JlI mounting solution (Dabco + Vectashield 1:1) in little drops
along the middle of the slide and apply a glass coverslip.
9. Store slides in the dark until signal analyzation with the fluorescence
microscope.
10. The slides can be stored at - 20
D C in the dark.
Signal enumeration
Microscope: For viewing the hybridization-signals, an epi-illumination
fluorescence microscope is required.
Light source: A 100 watt mercury lamp is the recommended excitation
light source for the Vysis probes.
Objectives: oil immersion fluorescence objectives with numeric apertures ) 0.75; a lOx objective, a 63x and a 100x oil immersion type objective are necessary for scanning and signal analysis.
Filters: multi-bandpass fIlters and single bandpass fluorescence microscope fIlter sets.
Enumeration scan: a minimum of 50 nuclei for each DNA probe
should be counted whenever possible and the percentage of aneuploid
signals should be determined.
Depending on the week of gestation in which amniocentesis was done,
you will find more or less nuclei showing hybridization signals. In the area
hybridized with the cosmid-DNA probes (LSI1312l) there are always less
nuclei showing good signals than in the area hybridized with the CEP 18/
X/Y probe. Therefore it is recommended to drop more suspension with
nuclei on this area of the slide.
Nuclei with diffuse and weak signals, split signals and very stringy signals will also be found. Counting these nuclei should be avoided. For the
analysis, only nuclei with clear and distinguishable signals should be used.
For further instructions to "signal enumeration" and "troubleshooting" we refer to the instructions from Vysis, which are included in
each AneuVysion EC kit.
123
6. Tip off the stain solution into a bottle for special waste disposal (DAPI
is a possible mutagen) and place the slides into a Coplin jar. Rinse 3
times with deionized water.
7. Let the slides dry in the dark.
8. Apply 20 JlI mounting solution (Dabco + Vectashield 1:1) in little drops
along the middle of the slide and apply a glass coverslip.
9. Store slides in the dark until signal analyzation with the fluorescence
microscope.
10. The slides can be stored at - 20
D C in the dark.
Signal enumeration
Microscope: For viewing the hybridization-signals, an epi-illumination
fluorescence microscope is required.
Light source: A 100 watt mercury lamp is the recommended excitation
light source for the Vysis probes.
Objectives: oil immersion fluorescence objectives with numeric apertures ) 0.75; a lOx objective, a 63x and a 100x oil immersion type objective are necessary for scanning and signal analysis.
Filters: multi-bandpass fIlters and single bandpass fluorescence microscope fIlter sets.
Enumeration scan: a minimum of 50 nuclei for each DNA probe
should be counted whenever possible and the percentage of aneuploid
signals should be determined.
Depending on the week of gestation in which amniocentesis was done,
you will find more or less nuclei showing hybridization signals. In the area
hybridized with the cosmid-DNA probes (LSI1312l) there are always less
nuclei showing good signals than in the area hybridized with the CEP 18/
X/Y probe. Therefore it is recommended to drop more suspension with
nuclei on this area of the slide.
Nuclei with diffuse and weak signals, split signals and very stringy signals will also be found. Counting these nuclei should be avoided. For the
analysis, only nuclei with clear and distinguishable signals should be used.
For further instructions to "signal enumeration" and "troubleshooting" we refer to the instructions from Vysis, which are included in
each AneuVysion EC kit.