244
HEINZ-ULRICH G. WEIER
2. Apply the hybridization mixture to the slide and coverslip.
3. Denature the slide at 88-92°C for 90 sec on a hot plate.
4. Transfer the slide to a moist chamber and incubate overnight at 37°C.
Washing and detection steps are not much different from protocols used
for FISH in interphase and metaphase cells that have been described in
detail (Weier et al. 1995, Duell et al. 1997, 1998; Wang et al. 1997):
1. After hybridization, wash the slide three times in 2x SSC at 20°C for
10 min each.
2. Incubated the slide with 100 III PNM buffer or blocking stock solution
under a plastic coverslip at 20°C for 5 min.
3. The slide is then incubated at room temperature for 30 min with 100 III
PNM buffer containing AMCA-avidin (Pharmacia), anti-digoxigeninrhodamine (Roche Molecular Biochemicals) and a mouse antibody
against FITC (DAKO).
Note: If only two labels are used, Le., biotin and digoxigenin, bound
probes are detected with avidin-FITC DCS (Vector) and anti-digoxigenin-rhodamine, respectively.
4. The slide is washed two to three times in 2xSSC for 15 min each at 20°C
with constant motion on a shaking platform.
5. If necessary, signals are amplified using a biotinylated antibody against
avidin raised in goat (Vector) followed by another layer of AMCA-avidin, a Texas Red-labeled antibody against sheep raised in rabbit (Sigma) and a horse-anti-mouse antibody conjugated to FITC (Vector)
(Wang et al. 1996).
6. The slide is mounted in 8 III of DAPI (0.05 Ilglrnl in antifade solution)
and covered by a 22x22 mm coverslip.
Subprotocol 8
Digital image acquisition and analysis
Although not a prerequisite for QDFM, digital image acquisition and computer-assisted analysis greatly facilitate the analysis of hybridization
images. Since QDFM is based on simple measurements of distances between probe hybridization domains, the analysis can alternatively be per-
HEINZ-ULRICH G. WEIER
2. Apply the hybridization mixture to the slide and coverslip.
3. Denature the slide at 88-92°C for 90 sec on a hot plate.
4. Transfer the slide to a moist chamber and incubate overnight at 37°C.
Washing and detection steps are not much different from protocols used
for FISH in interphase and metaphase cells that have been described in
detail (Weier et al. 1995, Duell et al. 1997, 1998; Wang et al. 1997):
1. After hybridization, wash the slide three times in 2x SSC at 20°C for
10 min each.
2. Incubated the slide with 100 III PNM buffer or blocking stock solution
under a plastic coverslip at 20°C for 5 min.
3. The slide is then incubated at room temperature for 30 min with 100 III
PNM buffer containing AMCA-avidin (Pharmacia), anti-digoxigeninrhodamine (Roche Molecular Biochemicals) and a mouse antibody
against FITC (DAKO).
Note: If only two labels are used, Le., biotin and digoxigenin, bound
probes are detected with avidin-FITC DCS (Vector) and anti-digoxigenin-rhodamine, respectively.
4. The slide is washed two to three times in 2xSSC for 15 min each at 20°C
with constant motion on a shaking platform.
5. If necessary, signals are amplified using a biotinylated antibody against
avidin raised in goat (Vector) followed by another layer of AMCA-avidin, a Texas Red-labeled antibody against sheep raised in rabbit (Sigma) and a horse-anti-mouse antibody conjugated to FITC (Vector)
(Wang et al. 1996).
6. The slide is mounted in 8 III of DAPI (0.05 Ilglrnl in antifade solution)
and covered by a 22x22 mm coverslip.
Subprotocol 8
Digital image acquisition and analysis
Although not a prerequisite for QDFM, digital image acquisition and computer-assisted analysis greatly facilitate the analysis of hybridization
images. Since QDFM is based on simple measurements of distances between probe hybridization domains, the analysis can alternatively be per-