134
ANITA RAUCH
Fig. 3. FISH on human sperm cells with two single copy probes from chromosome II labeled in red (r) and green (g) indicating euploidy for chromosome II
from diploidy, two color FISH and the simultaneous scoring of two different chromosomes are recommended. Moreover, in interphase, the FISH
obtained signal number varies artificially to a certain degree, because of
several factors discussed in the Comments section.
Troubleshooting
No or weak signals
Too much
background
Diffuse signals
and cells
Reasons for no or weak signals may be too short a denaturation or hybridization time or washing slide after hybridization with too high stringency. Stringency decreases with increase in the salt concentration or decrease in temperature and formamide concentration. Hybridization efficiency is increased by higher dextran sulfate and probe concentrations.
Too much background fluorescence may result from any soiling and can
be decreased during hybridization or posthybridization washing by increasing the temperature or formamide concentration, or decreasing
salt concentrations.
Decrease denaturation time or temperature by 2°C or shorten decondensation time.
ANITA RAUCH
Fig. 3. FISH on human sperm cells with two single copy probes from chromosome II labeled in red (r) and green (g) indicating euploidy for chromosome II
from diploidy, two color FISH and the simultaneous scoring of two different chromosomes are recommended. Moreover, in interphase, the FISH
obtained signal number varies artificially to a certain degree, because of
several factors discussed in the Comments section.
Troubleshooting
No or weak signals
Too much
background
Diffuse signals
and cells
Reasons for no or weak signals may be too short a denaturation or hybridization time or washing slide after hybridization with too high stringency. Stringency decreases with increase in the salt concentration or decrease in temperature and formamide concentration. Hybridization efficiency is increased by higher dextran sulfate and probe concentrations.
Too much background fluorescence may result from any soiling and can
be decreased during hybridization or posthybridization washing by increasing the temperature or formamide concentration, or decreasing
salt concentrations.
Decrease denaturation time or temperature by 2°C or shorten decondensation time.