- Centrifuge 2-5 mL of whole amniotic fluid (AF) specimen for 5 minutes at 1,000 RPM. The specimen should not appear bloody or brown.
- Resuspend the pellet in 2-5 mL of 1X trypsin/EDTA (0.05% Trypsin, 0.53mM EDTA 4Na in Hanks' Balanced Salt Solution without CaCl2, MgCl2 6H2O and MgSO4 7H2O) and incubate in a 37 ºC water bath for at least 15 minutes.
- Centrifuge the suspension for 5 minutes at 1,000 RPM.
- Resuspend the pellet in 2-5 mL of 0.56% KCl and incubate for 20 minutes in a 37 ºC water bath.
- Add 0.8-2 mL of fixative (3:1 methanol:glacial acetic acid) to the cells/hypotonic solution and vortex gently.
- Centrifuge the suspension for 5 minutes at 1,000 RPM and resuspend the pellet in 1 mL fresh fixative. Store fixed specimens at 4 ºC for at least 30 minutes or until ready to perform FISH. For long-term storage, store fixed specimens at -20 ºC in fixative.
- Before placing cells on slides, adjust volume of cell suspension according to size of cell pellet. If needed, particularly after a prolonged storage (>1 month), wash pellets with fixative before slide preparation.
- To prepare slides for FISH, drop the cell suspension directly onto 1 or 2 cold, glass slides, making 2 hybridization areas (15-25 µL of cell suspension per area).
Follow with FISH pretreatment kit