Fig 11 (FISH guide)
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LegendFig. 11.a-i)Comparative genomic hybridization (CGH).a) CGH with nick-translated probes, using insufficient amount of competitor DNA. Centromeres of many chromosomes yield a very bright signal. b) CGH with DOP-PCR labeled DNA. Hybridization showed less visible centromeres (only some acrocentrics) and regions of deletion and amplifications became visible. However, hybridization signals are more "dotty", doe to stringent (15 minutes, 65 C in 0.2x SSC) posthybridization washing conditions. c) Specific CGH hybridization signals, with no centromere hybridizations can be obtained when the appropriate amount of competitor DNA is taken. The smoothness of the signal is increased by using less stringent posthybridization washes, in 1-2x SSC at 42 C. d-i) Good CGH analysis can be obtained by labeling the DNA with either fluorescent labeled nucleotides, FITC and Rhodamine in "d", or with haptene labeled nucleotides, BIO and DIG in "g". The green and red channels of the "d" image (e, f) and the "g" image (h, i) are also shown. j-k)Multiplex FISH (M-FISH).j) Individual paint probes need to be tested for their uniformity in chromosome coverage. labeling procedure and nucleotides need also testing, separately and in combinations. Two red, two green and two blue chromosome painting probes were simultaneously tested. k) M-FISH on a normal metaphase with the Speicher approach, which uses a combination of five different fluorophores. The fluorophores used in this experiment were AMCA, FITC, Cy3, Cy3.5 and Cy5. l) FISH on paraffin embedded material (PET). Three chromosome 12p, band-specific, chromosome paint probes obtained by microdissection (depicted in Fig. 10l) were hybridized onto 10 micron PET sections from germ cell tumors (GCT) to identify regions of 12p amplifications, characteristic to the GCT. 5-6 12p copies were visible in the nuclei of the sample shown.