John M. S. Bartlett.pdf - Bio-Nica.info

460 Speel, Ramaekers, and Hopman
4. Wash the slides for 2 × 5 min with washing buffer and for 5 min with 1× PBS (all steps
in a Coplin jar).
5. Mount slides in Vectashield containing 0.5 µg/mLDAPI or 3000× diluted TOTO-3 iodide.
6. Examine the slides as described in Subheading 3.1.2., step 12.
3.3. Protocol 3
3.3.1. Immunofluorescence Detection of Structural Proteins in Chromosomes
1. Prepare metaphase spreads from human peripheral blood lymphocytes as described
previously (18,19).
2. After hypotonic treatment of the cell suspension for 10 min at 37°C in 75 mM KCl,
cytocentrifuge the cells onto alcohol-cleaned glass slides with 65–275g for 4 min and
air dry for 2 min.
3. Place slides in a Coplin jar containing KCM solution for 10 min at room temperature,
followed by incubation for 10 min at room temperature with 100 µL of blocking buffer
under a coverslip in a humid chamber.
4. Incubate slides for 30 to 45 min at room temperature with 50 µL of the primary antibody,
diluted in blocking buffer, under a coverslip in a humid chamber.
5. Wash slides for 2 × 5 min with KCM in a Coplin jar.
6. Incubate slides for 30 to 45 min at room temperature with 50 µL of FITC-conjugated
secondary antibody, diluted in blocking buffer, under a coverslip in a humid chamber
(see Note 4).
7. Wash slides for 2 × 5 min with KCM, and fix the chromosomes in fixation solution for 5
to 15 min at room temperature in a Coplin jar.
8. Wash slides in destilled water, airdry and store in the dark at room temperature until use.
3.2.3. PRINS DNA Labeling
1. To improve the signal intensity after PRINS labeling, the slides are immersed in 0.1 M NaOH
for 10 to 40 s (see Note 14) followed by neutralization with 2 × 5-min washes in 0.01 M
Tris-HCl, pH 7.4, and a wash in destilled water (all steps in a Coplin jar) before airdrying.
2. Fix slides in methanolacetic acid (31) for 2 × 2 min in a Coplin jar (see Note 15), wash
in 0.01 M Tris-HCl, pH 7.4, for 2 × 5 min in a Coplin jar, dehydrate slides in a series of
cold (4°C) 70, 96, and 100% ethanol, and airdry (see Note 16).
3. Denature chromosomal DNA in denaturation solution for 45 min at 4°C and neutralize in
0.01 M Tris-HCl, pH 7.4, for 2 × 5 min at room temperature (all steps in a Coplin jar).
4. Remove excess fluid by draining and blow slides dry with a jet of air.
5. Perform PRINS reaction, mounting and evaluation of the slides as described in Subheading
3.1.2., steps 2–12).
4. Notes
1. So far, specific oligonucleotide primers have been defined for the centromere regions
of up to 20 chromosomes (7,24,25). Particularly, the ability of primers to differentiate
between closely related sequences has made it possible to define α-satellite primers
for some chromosomes indistinguishable by FISH with centromeric probes, such as
for chromosomes 13 and 21, that only exhibit a one-base differerence at their 3′ end.
Furthermore, a number of chromosome-specific telomere primers have been generated, as
well as HPV-specific and single-gene-specific oligonucleotides (11,26,27).
2. Several companies commercialize specialized thermal cyclers with a flat block, including
Hybaid, Perkin–Elmer, Techne Corporation (Cambridge, UK), and MJ Research Inc.
(Watertown, MA). Because of differences in the design of the heating block, PRINS