250
HEINZ-ULRICH G. WEIER
applied to hybridization and probe detection on metaphase chromosomes,
this system works well with cDNA probes of several kb.
Troubleshooting
• Slide pretreatment
Slides from different manufacturers or even of the same brand may
produce very different qualities of fibers. Use a large batch of slides
from one manufacturer; avoid slides that are painted on one end, since
the paint might come off during pretreatment. Slides that have a sandblasted area at one end are preferable.
• Homogenous stretching of DNA molecules
Different procedures have been described to stretch DNA molecules. In
our hands, stretching involving a hydrodynamical force (meniscus) at
20°C or 4°C has proven most reproducible. There is, however, no need
to wait until the preparation has dried to completion. Once the DNA
molecules have bound to the substrate, the coverslip can be lifted to
exert the hydrodynamic stretching force (Hu et al. 1996).
• Immunocytochemical signal amplification
Never let the slides or part of them dry out during the immunocytochemical signal amplification. It is important to just drain the liquids
from the slides, and then rapidly apply the next solution such as a
blocking solution or the antibodies. If the slides are allowed to dry
out, the level of background staining will increase to unacceptable levels.
• Image acquisition
Most fluorochromes fade very quickly. Thus, minimize the exposure of
slides to the excitation light. Don't waste time studying the beauty of
the molecules.
• Image analysis
Always measure additional segments of the molecule such as the vector
segment, since these might provide additional information about the
extent and homogeneity of DNA stretching.
• Errors
Relative standard deviations are typically in the order of 5%. Higher
standard deviations provide a simple means of controlling the procedure. They signal it is necessary to check the data analysis results for
HEINZ-ULRICH G. WEIER
applied to hybridization and probe detection on metaphase chromosomes,
this system works well with cDNA probes of several kb.
Troubleshooting
• Slide pretreatment
Slides from different manufacturers or even of the same brand may
produce very different qualities of fibers. Use a large batch of slides
from one manufacturer; avoid slides that are painted on one end, since
the paint might come off during pretreatment. Slides that have a sandblasted area at one end are preferable.
• Homogenous stretching of DNA molecules
Different procedures have been described to stretch DNA molecules. In
our hands, stretching involving a hydrodynamical force (meniscus) at
20°C or 4°C has proven most reproducible. There is, however, no need
to wait until the preparation has dried to completion. Once the DNA
molecules have bound to the substrate, the coverslip can be lifted to
exert the hydrodynamic stretching force (Hu et al. 1996).
• Immunocytochemical signal amplification
Never let the slides or part of them dry out during the immunocytochemical signal amplification. It is important to just drain the liquids
from the slides, and then rapidly apply the next solution such as a
blocking solution or the antibodies. If the slides are allowed to dry
out, the level of background staining will increase to unacceptable levels.
• Image acquisition
Most fluorochromes fade very quickly. Thus, minimize the exposure of
slides to the excitation light. Don't waste time studying the beauty of
the molecules.
• Image analysis
Always measure additional segments of the molecule such as the vector
segment, since these might provide additional information about the
extent and homogeneity of DNA stretching.
• Errors
Relative standard deviations are typically in the order of 5%. Higher
standard deviations provide a simple means of controlling the procedure. They signal it is necessary to check the data analysis results for