Procedure
Slide Preparation
Hybridization
Detection
Slide Preparation
- Start with chromosome preparations from any cell type.
- Incubate with 200 µL RNase for 1 hour at 37 °C
- Wash slides in 2x SSC for 5 minutes. Repeat.
- Rinse slides in 10 mM HCl.
- Incubate with 200 µL pepsin for 10 minutes at 37 °C.
- Rinse slides in deionized H2O.
- Wash slides in 2x SSC for 5 minutes. Repeat.
- Stabilize slides in paraformaldehyde for 10 minutes.
- Wash slides in 2x SSC for 5 minutes. Repeat.
- Dehydrate slides in an ethanol series: 70%, 80%, 95%; 2 minutes each.
- Air dry.
Hybridization
- Prepare 30 µl hybridization solution per slide. Heat to 70 °C. for 10 minutes and place on ice.
- Place 30 µl of hybridization solution on each slide and cover with a plastic cover slip.
- Denature slide at 65-70 °C for 5 minutes on heat block.
- Gradually decrease temperature to 37 °C.
- Hybridize at 37 °C overnight in humidity chamber.
Detection
- Wash slides in 2x SSC to remove coverslip.
- Wash slides in wash buffer at 40 °C for 5 minutes. Repeat.
- Wash slides in 0.1x SSC at 40 °C for 5-15 minutes.
- Wash slides in 2x SSC at 40 °C for 5-15 minutes.
- Cool slides to room temperature.
- Equilibrate slides in detection buffer for 5 minutes.
- Block in blocking buffer for 20-30 minutes.
- Incubate with 50 µl antibody or detection compound for 30-60 minutes (e.g., 5 µg/ml Streptavidin-Cy3 in blocking buffer).
- Wash slides in 2x SSC for 5 minutes. Repeat twice.
- Counterstain with DAPI solution for 10 minutes.
- Rinse briefly and mount in antifade mounting medium.
- Analyze with fluorescence microscope.