8 Amniotic Fluid Cells - Uncultured
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7. Before placing the cells on slides, adjust volume of cell suspension according to the size of the pellet; if the pellet is very small, reduce the
suspension to a few drops (2-4) out of a blue or better a yellow tip.
8. Take the cold wet slide, let the water run off and place drops onto the
slide making two hybridization areas or use two slides with one hybridization area on each for differential post-hybridization washes (as described in outline).
Note: In the case of a small pellet which can hardly be seen, it is better
to drop the suspension on a nearly dry cold slide and to make an unequal distribution: the hybridization area for the LSI 13/21 probe
should contain more nuclei than the area for CEP 18/X/Y probe.
9. Dry the slide(s) for ca. 20 min at about 60°C.
Slide pretreatment
1. Place slide(s), prepared from uncultured amniocytes, in 1xPBS at
room temperature (RT) for 10 min.
2. Transfer slide(s) to freshly made pepsin working solution for 3-10 min
(depending on the density of nuclei on the slide and on the batch of
pepsin) at 37°C.
3. Rinse the slide(s) in 1xPBS/MgCl z at RT for 5 min.
4. Place the slides in post fixation solution (l xPBS/MgClz/formaldehyde)
for 10 min at RT. (After use, this solution should be poured into a special waste disposal).
5. Rinse the slides in 1xPBS at RT for 5 min.
6. Dry slides.
7. Immerse slide(s) in 70% ethanol for 5 min, then remove slides and
place in the 90% ethanol for 5 min, followed by 100% ethanol for 5 min.
8. Dry the slides for about 20 min.
Fluorescence in situ hybridization procedure
1. Prewarm the 74°C water bath (with a steel-plate inside for laying the Co-denaturation
slides on it) or a slide warmer.
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7. Before placing the cells on slides, adjust volume of cell suspension according to the size of the pellet; if the pellet is very small, reduce the
suspension to a few drops (2-4) out of a blue or better a yellow tip.
8. Take the cold wet slide, let the water run off and place drops onto the
slide making two hybridization areas or use two slides with one hybridization area on each for differential post-hybridization washes (as described in outline).
Note: In the case of a small pellet which can hardly be seen, it is better
to drop the suspension on a nearly dry cold slide and to make an unequal distribution: the hybridization area for the LSI 13/21 probe
should contain more nuclei than the area for CEP 18/X/Y probe.
9. Dry the slide(s) for ca. 20 min at about 60°C.
Slide pretreatment
1. Place slide(s), prepared from uncultured amniocytes, in 1xPBS at
room temperature (RT) for 10 min.
2. Transfer slide(s) to freshly made pepsin working solution for 3-10 min
(depending on the density of nuclei on the slide and on the batch of
pepsin) at 37°C.
3. Rinse the slide(s) in 1xPBS/MgCl z at RT for 5 min.
4. Place the slides in post fixation solution (l xPBS/MgClz/formaldehyde)
for 10 min at RT. (After use, this solution should be poured into a special waste disposal).
5. Rinse the slides in 1xPBS at RT for 5 min.
6. Dry slides.
7. Immerse slide(s) in 70% ethanol for 5 min, then remove slides and
place in the 90% ethanol for 5 min, followed by 100% ethanol for 5 min.
8. Dry the slides for about 20 min.
Fluorescence in situ hybridization procedure
1. Prewarm the 74°C water bath (with a steel-plate inside for laying the Co-denaturation
slides on it) or a slide warmer.