27 Yeast FISH: Delineation of Chromosomal Targets in Vegetative and Meiotic Yeast Cells
341
Subprotocol 3
Detection of hybrid molecules
Procedure
We present an indirect detection system for two colors, since only these
render readily distinguishable signals (Fig. 1) which are requisite for the
visual analysis of a large number of nuclei and hybridization patterns in
e.g. time course experiments.
1. Preparations are blocked against unspecific binding by placing them
5 min in a Coplin jar containing 0.5% BSA/BT buffer at 42°C.
2. Remove slides from Coplin jar, briefly drain excess fluid and place
them in a moist box. (Preparations should never dry up during this
and subsequent steps).
3. Apply 100 III BT buffer containing avidin-FITC (2.5 Ilglml) and antidig-rhodamine Fab fragments at 0.5 Ilglml.
4. Cover with large cover slip and incubate for 1 h at 42°C.
5. Gently slide down or float off coverslips in BT buffer.
6. Wash slides 3x3 min in BT buffer at 42°C.
7. Apply 100 III BT buffer containing biotinylated goat anti-avidin antibody (2 Ilglml).
8. Cover with cover slip and incubate for 30 min at 42°C in a moist chamber.
9. Wash 3x3 min in BT buffer at 42°C.
10. Repeat step 3 with avidin-FITC only and incubate for a final 30 min at
42°C.
11. Repeat step 6, remove slides from Coplin jar and briefly drain excess
fluid.
12. Apply 18 III antifade solution containing DAPI as a DNA-specific counterstain and mount a 24x60 mm coverslip.
13. Place slides in a drying block or under a paper towel. Apply gentle
pressure to remove excess liquid and trapped air bubbles.