Figure 2 (slideprep)
Fig. 2. a-c:Simultaneous denaturing using hybridization buffer containing 12-15% dextran sulfate and different brands of formamide. Slides were chemically aged 10 seconds at 94? C on a thermocyler block and DAPI stained. Use of dextran-sulfate during denaturing increases chromosome thickness and alters their structure. Formamide brand influences the process as well, with chromosomes being either excessively thick (a) or with a distorted, uneven surfaces (b). Among the three images, the best brand of formamide was in (c), with chromosomes not as thick as (a) and more evenly stained than (b).d:Another slide from the same lot as above was subjected to separate denaturing for 2 minutes in a Coplin jar, containing 70%FA/2xSSC at 75? C (same formamide as in Fig. 2c). The absence of dextran sulfate results in less distorted chromosomes. However, the brightly stained centromeres indicate over-denaturing of the chromosomes, yielding a pseudo C-banding aspect. Also compare with (g), where separate denaturing using the same formamide brand was done gradually, on the metal block of a thermocycler.e-f:10 minute hybridization of a probe for the alpha satellite repeat of chromosome 1, on cells chemically aged for 2 minutes. Arrows indicate weaker hybridization signals in (e) (no pepsin treatment) than in (f) (pepsin treated).g-h:20 minutes hybridization of a biotinylated commercial probe for chromosome 1 (Oncor), detected with avidin FITC (g). Slide was subjected to 10 seconds chemical aging and separate denaturing on the metal block of a thermocycler. The grayscale DAPI image of the metaphase shown in (g) was inverted using Adobe Photoshop (h). Chromosomes 1, 5 and 19 (carrying hybridization signals) are identifiable. i: 10 second chemical aging and separate slide denaturing on the metal block of a thermocycler followed by 5 hours hybridization using a commercial diagnosis probe (Oncor, Inc) for Prader-Willi syndrome.j-k: 10 seconds chemical aging and simultaneous denaturing of a CGH analysis on the metal block of a thermocycler followed by 18 hours hybridization. Normal DNA was digoxigenin-labeled and detected by antidigoxigenin-FITC antibody (j). Tumor DNA (testicular germ cell tumor) was biotin-labeled and detected with Avidin Cy3 (k). Arrows show the typical 12p amplification characteristic for this tumors. Although the hybridization was even and of good quality, note the thickness of the chromosomes, mostly due to the presence of the dextran sulfate in the hybridization buffer during the direct denaturing phase.l:10 seconds chemical aging and separate slide denaturing on a metal block of a thermocycler followed by 18 hours hybridization of the FITC-labeled painting probes of an M-FISH analysis.
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Last modified on: Feb12, 2001