29 Spectral Imaging Analysis of Metaphase and Interphase Cells
371
6. Mark a small circle on the back of an ethanol-cleaned slide using a
diamond tip pen.
7. Place hypotonic treated blastomeres on slides.
8. Fix the blastomeres with one drop of methanoUacetic acid (3/1 or 1/1,
voUvol).
9. Check the nucleus under a phase contrast microscope and mark its
position.
10. Dehydrate slides in three consecutive baths of 70%, 80% and 100%
ethanol for 2 min each.
11. Slides are now ready for FISH or can be stored at -20°C.
Amniocytes were donated for research under an approved protocol in accordance with guidelines set by the UCSF Committee on Human Research.
Uncultured amniocytes were obtained from 1-2 ml of amniotic fluid and
fixed on slides within a few hours of amniocentesis.
I. Pellet the cells and resuspend the cells in a hypotonic solution consisting of 0.3% KCI for 30 min at 37°e.
2. Add several drops of freshly made ice-cold fixative (methanoUacetic
acid, 3/1, voUvol) and gently mix.
3. Pellet the cells and resuspend the cells with ice-cold fixative several
times.
4. Drop the cells on fixative-cleaned slides above a boiling water bath.
5. Air dry the slides.
6. Check the nuclei on slides under a phase contrast microscope.
7. Incubate slides in 2x SSC at 37°C for 1 hr.
8. Pretreat slides with pepsin (50 Ilg/ml pepsin in 0.01 M HCI) at 37°C for
13 min.
9. Wash slides in 1x PBS at room temperature for 5 min.
10. Incubate slides in 1% formaldehyde in 1x PBS/MgCh at room temperature for 5 min.
II. Wash slides in lxPBS at room temperature for 5 min.
12. Dehydrate slides in 70%, 80%, and 100% ethanol for 2 min each.
13. Slides are now ready for FISH or can be stored at -20°e.
Uncultured human
amniocytes
371
6. Mark a small circle on the back of an ethanol-cleaned slide using a
diamond tip pen.
7. Place hypotonic treated blastomeres on slides.
8. Fix the blastomeres with one drop of methanoUacetic acid (3/1 or 1/1,
voUvol).
9. Check the nucleus under a phase contrast microscope and mark its
position.
10. Dehydrate slides in three consecutive baths of 70%, 80% and 100%
ethanol for 2 min each.
11. Slides are now ready for FISH or can be stored at -20°C.
Amniocytes were donated for research under an approved protocol in accordance with guidelines set by the UCSF Committee on Human Research.
Uncultured amniocytes were obtained from 1-2 ml of amniotic fluid and
fixed on slides within a few hours of amniocentesis.
I. Pellet the cells and resuspend the cells in a hypotonic solution consisting of 0.3% KCI for 30 min at 37°e.
2. Add several drops of freshly made ice-cold fixative (methanoUacetic
acid, 3/1, voUvol) and gently mix.
3. Pellet the cells and resuspend the cells with ice-cold fixative several
times.
4. Drop the cells on fixative-cleaned slides above a boiling water bath.
5. Air dry the slides.
6. Check the nuclei on slides under a phase contrast microscope.
7. Incubate slides in 2x SSC at 37°C for 1 hr.
8. Pretreat slides with pepsin (50 Ilg/ml pepsin in 0.01 M HCI) at 37°C for
13 min.
9. Wash slides in 1x PBS at room temperature for 5 min.
10. Incubate slides in 1% formaldehyde in 1x PBS/MgCh at room temperature for 5 min.
II. Wash slides in lxPBS at room temperature for 5 min.
12. Dehydrate slides in 70%, 80%, and 100% ethanol for 2 min each.
13. Slides are now ready for FISH or can be stored at -20°e.
Uncultured human
amniocytes