Fig 9 (FISH guide)
LegendFig. 9.a)Non-pretreated slides.Image shows the thin "film" of material covering the slide surface when slides are aged (particularly in dry heat) and are not pretreated with a protease. This "film" greatly impairs hybridization efficiency. b)Type 1 background (on nuclei and chromosomes). A well labeled and verified YAC probe (red) was hybridized using less than optimal competitor DNA amount. The specific signal was visible, but hard to identify among the background signals. c-d) Type 2 background (among and around nuclei and chromosomes - where the cytoplasmic residua is located); Same YAC probe was labeled again, but leaving the labeled fragments longer (above 5-600 bp average). Also, a somewhat lower quality cell suspension was used. c) The probe signal (red) is non-specific. For better contrast, YOYO was used as a counterstain; d). On a similar slide, the same YAC probe as in (c) but detected green was combined with a PAC probe, optimally labeled and cut (red). Even on lower quality slides, signals can be obtained if the probe is digested into small fragments (~ 200 bp average); e) Type 3 background (on the glass surface but not on nuclei and metaphases). Due to insufficient posthybridization wash or to a too high antibody concentration. In the image presented, signals from a chromosome 1 centromere probe are visible. f-g)Nick translation vs. PCR labeling. Same 3kb plasmid probe on 7q was labeled by both procedures, hybridized together with the centromere probe and detected with a red fluorophore (arrows). The PCR labeled probe(f) yielded somewhat stronger signals but with a higher background than the nick translated probe (g). h) Small PCR fragment hybridization. Two loci on chromosome Y (sY14 = 475bp and sY81 = 206 bp) were PCR amplified and labeled, then hybridized and detected with a red fluorophore. Signals from both fragments were visible (arrows) in over 50% of the metaphases but the background was high. Identification of the hybridization signals without prior information about their chromosome location would have been almost impossible. i) FISH on G-banded chromosomes. A 10 kb probe (red) was hybridized onto previously G-banded chromosomes, to determine its chromosome location. Although, for G-banding, the slide was aged at 65 C overnight, the denaturing step made chromosome more "puffy" and partially changed the position of some chromosomes (arrow). j-k) Signal amplification. A green and a red labeled cosmids were hybridized together on the same slide. The weak FITC signal (j) was amplified by using an anti-FITC antibody. Green signals were significantly improved (k). l)Combinatorial labeling. Three centromere probes, one labeled with a green fluor, one with a red fluor and one with both the green and red fluors (orange), were hybridized together (a nucleus is shown, no counterstain). Combinatorial labeling allowed detection of three probes using only two fluorophores.
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Last modified on: Feb12, 2001