Fig 10 (FISH guide)
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LegendFig. 10.a-c)Triple color FISH advantages.In each image, three probes (centromeres in "a", cosmid sets in "b" and "c") were labeled with three different fluorophores. a) chromosome 14/22 probe (green) was combined with the X (blue) and Y (red) centromeres which allow sex diagnosis; b) interphase position three cosmids relative to one another (interphase mapping); c) chromosomal position of three cosmids relative to one another (metaphase/chromosome mapping). In each case (a-c) triple color FISH is shorter and more informative than double color FISH. d) Combination of commercial probes. A commercially labeled chromosome paint probe (BIO-labeled) was combined with a dual-color cosmid probe combination (FITC and Rhodamine labeled, commercially available). The three probes were combined in the same vial, precipitated and hybridized together. BIO was detected with Cy5 and chromosomes were counterstained with DAPI and pseudocolored dark-yellow. All three probes worked after combination, although were not used according to the protocols provided by the two vendors. e)InterAlu PCR amplification of two YACs, one labeled red and one green. 4-5 times more green probe was used. The red signals are specific (arrows) but it is very difficult to identify the green specific signals from the pseudobanding pattern provided by the green probe. The amount of competitor DNA was appropriate for the red probe but not for the green probe. f) Nick translation labeling of a chimeric YAC. A chimeric YAC was labeled red by nick translation. Th appropriate amount of Cot1 DNA prevented repetitive fragments binding and provided good signals. The specific hybridization signal of this YAC was accompanied by a smaller red signal (arrows) on a different chromosome pair, indicating YAC chimerism. g) 20 bp primer hybridization. An Alu-specific, BIO-labeled primer was hybridized onto chromosomes, followed by gentle posthybridization washes and green detection. The hybridization signal is uniform and does not show the characteristic pseudo-banding pattern (compare with "e"). The very short primer probably finds many more targets than the longer fragments used for hybridization in "e". h-i)Tyramide detection (TSA). The TSA procedure utilizes an enzymatic reaction to precipitate many labeled tyramide molecules at the site of antibody binding and thus increases FISH signal. A BIO-labeled centromere probe for chromosome 1 was detected with Avidin FITC (h) or with the tyramide system (i). Signals are stronger in "i". j) Fiber FISH mapping. Two cosmid probes (35-40kb each), one labeled red and one green, were mapped at 40-50 kb from one another using halo-FISH (a variation of the fiber FISH procedure). k-l)Combination of complex FISH probes. Microdissected probes for the p and q arm of chromosome 12 were hybridized together with chromosome 12 centromere probe, and each was detected with a different color/fluor (k). Microdissected probes from the three subbands of chromosome 12p were labeled, each with a different fluorophore, and hybridized together (l). Virtually any probe, complex or less complex, can be combined with any other one if labeling combinations are carefully chosen.