104
ANITA RAUCH
1. Carefully remove rubber cement and coverslip with forceps.
2. Place slide in a Coplin jar with 1x SSC in a 75°C water bath for 2 min,
agitate the slide for the first 1-3 s.
3. Wash slide in 2xSSC/0.l% NP-40 at room temperature for 5 s-1 min.
Counterstaining
Storage
1. Place 1 ml DAPIIpropidium iodide working solution on each wet slide
and incubate for 5 min in the dark.
2. Place slide shortly in 2x SSC/O.l % NP-40 and air dry slide in the dark.
3. Apply 20 JlI of antifade in a row of dots across the slide and cover with a
coverslip.
4. Store slide at -20°C in the dark.
Results
Analyze slides with a suitable filter set under a fluorescence microscope.
According to Hook 1977 it is necessary to analyze at least 299 cells to exclude a 1% mosaicism with a confidence limit of 95%. However, in interphase FISH, the signal number obtained, varies artificially to a certain degree, because of several factors which are discussed in the Comments section. Therefore it is necessary to analyze a larger number of nuclei and
interpret the results based on statistics from normal controls. With directly labeled probes from VYSIS Corp., the following results were
achieved. For normal controls, the mean percentage of cells with a normal
signal pattern on buccal smears was 99% for the repetitive centromeric
probe CEP X and 98% for the single copy probes LSI 21 and 20q13. In
normal controls of urinary sediment, a normal signal pattern of LSI
20q13 was found in 98.7% of cells (see Table 1). If a threshold standard
deviation of 4 is considered sufficient, this method would detect trisomic
cell lines constituting at least 1.4% (CEP) and 2% (LSI) of the total cell
population and monosomic cell lines constituting at least 2.5% (CEP)
and 4% (LSI). However, for diagnostic purposes, each lab has to set up
their own normal values and a second DNA probe should be cohybridized
as an internal control of hybridization efficiency.
ANITA RAUCH
1. Carefully remove rubber cement and coverslip with forceps.
2. Place slide in a Coplin jar with 1x SSC in a 75°C water bath for 2 min,
agitate the slide for the first 1-3 s.
3. Wash slide in 2xSSC/0.l% NP-40 at room temperature for 5 s-1 min.
Counterstaining
Storage
1. Place 1 ml DAPIIpropidium iodide working solution on each wet slide
and incubate for 5 min in the dark.
2. Place slide shortly in 2x SSC/O.l % NP-40 and air dry slide in the dark.
3. Apply 20 JlI of antifade in a row of dots across the slide and cover with a
coverslip.
4. Store slide at -20°C in the dark.
Results
Analyze slides with a suitable filter set under a fluorescence microscope.
According to Hook 1977 it is necessary to analyze at least 299 cells to exclude a 1% mosaicism with a confidence limit of 95%. However, in interphase FISH, the signal number obtained, varies artificially to a certain degree, because of several factors which are discussed in the Comments section. Therefore it is necessary to analyze a larger number of nuclei and
interpret the results based on statistics from normal controls. With directly labeled probes from VYSIS Corp., the following results were
achieved. For normal controls, the mean percentage of cells with a normal
signal pattern on buccal smears was 99% for the repetitive centromeric
probe CEP X and 98% for the single copy probes LSI 21 and 20q13. In
normal controls of urinary sediment, a normal signal pattern of LSI
20q13 was found in 98.7% of cells (see Table 1). If a threshold standard
deviation of 4 is considered sufficient, this method would detect trisomic
cell lines constituting at least 1.4% (CEP) and 2% (LSI) of the total cell
population and monosomic cell lines constituting at least 2.5% (CEP)
and 4% (LSI). However, for diagnostic purposes, each lab has to set up
their own normal values and a second DNA probe should be cohybridized
as an internal control of hybridization efficiency.