27 Yeast FISH: Delineation of Chromosomal Targets in Vegetative and Meiotic Yeast Cells
335
2. Spin down cells and dissolve pellet in lho volume absolute ethanol with
respect to the starting aliquot (see procedure 1.2).
3. Distribute 20 III of ethanol-fIxed nuclei uniformly on a clean glass slide
by streaking out with one edge of a cover slip without scratching along
the surface of the slide.
4. Dry down the preparation on a heating block at 60°C for 10-15 min.
5. Apply 18 III of antifade solution containing DAPI as a DNA specifIc dye
and examine the density of nuclei under a fluorescence microscope. If
required, remove cover slip, wash in dH 2 0, air dry, then add more
ethanol fIxed spheroplasts to the slide and allow them to adhere
(see above).
6. To open up fIxed chromatin, incubate preparations for 1 h-overnight
in 4xSSC/0.5% Tween 20/pre-boiled RNaseA (1 Ilglml) at 60°C
(Scherthan et al. 1993).
7. Slide down coverslip, wash slides at room temperature for 5 min in
PBS.
8. Digest remaining RNA for >60 min at 37°C by adding 100 III of preboiled RNaseA at a concentration of 200 /lg/ml 2xSSC under a large
coverslip. Use a moist chamber for incubation.
9. Wash preparations once in PBS and drain off excess fluid.
1.5 Preparation of nuclear spreads
Since undisrupted yeast nuclei generally display a small diameter (2-4 /lm)
the technique of detergent spreading may be used to enhance cytological
resolution in the yeast nucleus (Dresser and Giroux 1988, Loidl et al. 1991,
Jin et al. 1998). Depending on the mode of pre-fIxation (see procedure 1.2,
step 6), it is possible to control the degree of nuclear spreading and to
preserve the relative morphology of nuclei during spreading (TrellesSticken et al. 1999).
Limited nuclear spreading may be applied to obtain moderately enlarged
nuclei with relatively preserved nuclear architecture.
1. Subject cells to pre-ftxation as described under procedure 1.1 and
spheroplast them as far as procedure 1.2, step 7.
Limited detergent
spreading of nuclei
335
2. Spin down cells and dissolve pellet in lho volume absolute ethanol with
respect to the starting aliquot (see procedure 1.2).
3. Distribute 20 III of ethanol-fIxed nuclei uniformly on a clean glass slide
by streaking out with one edge of a cover slip without scratching along
the surface of the slide.
4. Dry down the preparation on a heating block at 60°C for 10-15 min.
5. Apply 18 III of antifade solution containing DAPI as a DNA specifIc dye
and examine the density of nuclei under a fluorescence microscope. If
required, remove cover slip, wash in dH 2 0, air dry, then add more
ethanol fIxed spheroplasts to the slide and allow them to adhere
(see above).
6. To open up fIxed chromatin, incubate preparations for 1 h-overnight
in 4xSSC/0.5% Tween 20/pre-boiled RNaseA (1 Ilglml) at 60°C
(Scherthan et al. 1993).
7. Slide down coverslip, wash slides at room temperature for 5 min in
PBS.
8. Digest remaining RNA for >60 min at 37°C by adding 100 III of preboiled RNaseA at a concentration of 200 /lg/ml 2xSSC under a large
coverslip. Use a moist chamber for incubation.
9. Wash preparations once in PBS and drain off excess fluid.
1.5 Preparation of nuclear spreads
Since undisrupted yeast nuclei generally display a small diameter (2-4 /lm)
the technique of detergent spreading may be used to enhance cytological
resolution in the yeast nucleus (Dresser and Giroux 1988, Loidl et al. 1991,
Jin et al. 1998). Depending on the mode of pre-fIxation (see procedure 1.2,
step 6), it is possible to control the degree of nuclear spreading and to
preserve the relative morphology of nuclei during spreading (TrellesSticken et al. 1999).
Limited nuclear spreading may be applied to obtain moderately enlarged
nuclei with relatively preserved nuclear architecture.
1. Subject cells to pre-ftxation as described under procedure 1.1 and
spheroplast them as far as procedure 1.2, step 7.
Limited detergent
spreading of nuclei