6 In Situ Hybridization for DNA: Fluorescent Probe
81
Follow step 22.
23. Remove a slide from PN buffer, drain and blot excess liquid
form edge.
24. Apply diluted anti-digoxigenin-FITC or -TexasRed (10-20
times diluted in PNM buffer, Sill! cm 2 ), and cover with a plastic coverslip cut from a polyethylene bag.
25. The slide is then incubated for 20 mins in a moist chamber at
room temperature in darkness.
26. Wash the slides in 3 changes ofPN buffer at room temperature for 2 mins each.
27. Mount in an anti-fade solution (1.51ll!cm 2 ) containing DAP,
or PI for FITC, and a no. 1 thickness coverslip used. View
using fluorescent microscope.
Results
Tandem repeats in the centromeric regions of all chromosomes
are specific for each chromosome. The probes for these sequences identify each chromosome and detect numerical aberrations in the nuclei. Hybridization signals are compact in interphase nuclei. Furthermore, the target sequences are very bright,
since there are frequently megabases of target for short probe
sequences (Fig. 1, 2). The second type of probe makes one or
more whole chromosomes or chromosomal segments visible,
and can be used to analyze structural abnormalities in metaphase spreads (Fig.3). The third type of probe is specific for genome loci and can be used to detect genetic events such as deletions, amplifications, inversions, and translocations in interphase nuclei (Fig. 4,5).
Fluorescent
staining using
digoxigenin
labelled
probes
81
Follow step 22.
23. Remove a slide from PN buffer, drain and blot excess liquid
form edge.
24. Apply diluted anti-digoxigenin-FITC or -TexasRed (10-20
times diluted in PNM buffer, Sill! cm 2 ), and cover with a plastic coverslip cut from a polyethylene bag.
25. The slide is then incubated for 20 mins in a moist chamber at
room temperature in darkness.
26. Wash the slides in 3 changes ofPN buffer at room temperature for 2 mins each.
27. Mount in an anti-fade solution (1.51ll!cm 2 ) containing DAP,
or PI for FITC, and a no. 1 thickness coverslip used. View
using fluorescent microscope.
Results
Tandem repeats in the centromeric regions of all chromosomes
are specific for each chromosome. The probes for these sequences identify each chromosome and detect numerical aberrations in the nuclei. Hybridization signals are compact in interphase nuclei. Furthermore, the target sequences are very bright,
since there are frequently megabases of target for short probe
sequences (Fig. 1, 2). The second type of probe makes one or
more whole chromosomes or chromosomal segments visible,
and can be used to analyze structural abnormalities in metaphase spreads (Fig.3). The third type of probe is specific for genome loci and can be used to detect genetic events such as deletions, amplifications, inversions, and translocations in interphase nuclei (Fig. 4,5).
Fluorescent
staining using
digoxigenin
labelled
probes