120
RENATE ULMER
Preparing
the solutions
Table 1.
ethanol series
hypotonic solution
1 M HCI
1M MgCIz
20xSSC
IOxPBS
lxPBS
1x PBS/MgCIz
1x PBS/MgCI 2 1
formaldehyde
pepsin working solution
mounting solution
Procedure
70%,90 %,100 %
10 ml 1% sodium citrate 15 ml 0.33% MgCI2 1 ml hyaluronidase (150 U) alternatively: 0.28% KCI solution
8.62 ml 32% HCI 91.38 ml purified water
101.65 g MgCIz x 6 H 2 0 add distilled water to a final
volume of 500 ml
175.3 g NaCI 88.2 g sodium citrate 800 ml distilled water
adjust to pH 7.0 add distilled water to a final volume of
1000 ml
98.4 g PBS (powder) 1000 ml distilled water
40 ml 10xPBS 360 ml distilled water
190 ml PBS 10 ml 1 M MgCI2
100 ml PBS/MgCIz 3 ml paraformaldehyde
50 III of 10% pepsin solution 100 ml distilled water 1 ml
1M HCI
Dabco + Vectashield mixed 1:1
Slide preparation from uncultured amniotic fluid
1. Put clean slides (cleaned for example with tissue paper) into a Coplin
jar with purified water. Place into the refrigerator.
2. Centrifuge 3-5 ml of whole amniotic fluid (AF) specimen for 5 min at
1000 rpm.
3. Resuspend the pellet in 2-5 ml hypotonic solution (37°C) and incubate
for 20 min at 37°C. (In order to lose as few cells as possible, use a pipette with a plastic tip or a siliconized pipette for resuspending the pellets).
4. For prefixation add 0.5-1 ml cold fixative and mix gently.
5. Centrifuge the suspension for 5 min at 1000 rpm.
6. Resuspend the pellet in 1-2 ml fresh fixative, centrifuge again at 1000
rpm and resuspend the pellet again in fresh fixative. After 5 min or
longer, centrifuge one last time at 1000 rpm for 5 min.