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Reviewer Comments

Reviewed by: Luis Antonio Parada, CIC Biogune, Derio, Spain.

1. In my experience the primers are better preserved when kept at higher concentration (100µM) and different volumes are added according to the required concentrations. Usually the amplification products (unlabeled) are purified by precipitation with ethanol and dissolved in bi-distilled water at high concentration to preserve the DNA. The concentrations of plain and fluorescent nucleotides I use for the DOP PCR labeling reaction are lower than the used by this group without compromising the quality and efficiency of the painting probes. For example the final concentrations are as follow: dNTP=10µM; dTTP=7µM; dTTP-Spectrum Orange=50µM; dTTP-Biotin=30µM; dTTP-Digoxigenin=30µM.

2. I routinely assemble the nick translation reaction on ice. More importantly the working dilution of DNaseI is also prepared with cold water and kept on ice until the moment just before starting the reaction. Apart from adding 1µl of EDTA 0.5M, I also heat the sample at 65°C for 10 minutes to stop the reaction.

3. The concentration of the cell suspension varies according to the size of the coverslips used. Usually 10⁵ cells resuspended in 50µl of PBS are enough per 13mm diameter round coverslip. Whereas 2-4x10⁵ cells resuspended in 250µl of PBS suffice for one 22x22mm coverslip.

4. In addition to digesting the paraffin embedded tissue sections with Pepsin, pretreatment with 10µg/ml proteinase-K in 1xPBS at 37°C (after washing the pepsin in water) improves the penetration of DNA probes, especially whole chromosome painting probes.

5. Excessive drying of DNA during the precipitation procedure of FISH probes may reduce the solubility of the DNA in the hybridization mix. The probe can also be dissolve by incubating the DNA in an appropriate volume of Formamide 100% pH 7.2 at 37°C, for 30 minutes with strong agitation (vortex) and short centrifugation every 10 minutes. Then equal volume of dextran sulfate 20%/4XSSC is added to reach the appropriate concentration of DNA in a final hybridization cocktail constituted of 50% of Formamide, 2XSSC and 10% dextran sulfate.

6. Hybridization of the DNA probe to the target DNA can also be performed in incubators at 37°C using humid hybridization chambers prepared with large Petri dishes or in staining boxes with light protective tight lid. Denaturation of painting probes addressed to two or three chromosomes (Double or triple painting) can be performed at 80°C for 10 minutes and then pre-annealed 30 minutes at 37°C without damaging the probe. Denaturation of target DNA of tissue sections often requires 5-7 minutes at 75°C. In all cases immersion of the cover slips or slides  in ice cold  50% formamide/2XSCC  stops the denaturation process and make the target DNA ready for hybridization.

7. The stringency of the washing solutions during the detection procedure may vary with the different type o, f DNA probes.  For example:
   Chromosome painting and BAC clones probes 
   1x5 minutes. 50% formamide/2xSSC at 45°C
   1x5 minutes. 1xSSC at 45°C
   1x5 minutes. 4xSSC/0.1% Tween 20 at 45°C
   1x5 minutes. 4xSSC/0.1% Tween 20 at RT
   Pancentromeric probes
   1x5 minutes. 50% formamide/2xSSC at 60°C
   1x5 minutes. 0.1xSSC at 60°C
   1x5 minutes. 4xSSC/0.1% Tween 20 at 60°C
   1x5 minutes. 4xSSC/0.1% Tween 20 at RT.

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Figures

Figure 1: 3 colour 3D-FISH on nuclei of normal diploid human fibroblasts. Maximum intensity projections of confocal serial sections are shown. Chromosome territories3 (green; DNP = detected with FITC), 5 (blue; Dig = detected with Cy3), and 11 (red; Bio = detected with Cy5). Click here for a larger view.

Figure 2: The gel shows typical patterns after DOP2 (upper row) and DOP3 (bottom row) amplification of the genomic DNA of ten different BAC clones. Proper amplification typically yields a DNA smear between 0.3-4kb. Note within the smear for each BAC fragments of higher intensity which are not the same in DOP2 and DOP3 amplifications for the corresponding BACs.

Figure 3: The gel shows typical patterns after DOP2 (upper row) and DOP3 (bottom row) label amplification of primary DOP2/DOP3 amplification prodcts of ten different BAC clones (same as in protocol 4). Each lane was loaded with 2µl label amplification product. In contrast to the primary amplification the label PCR results in shorter fragments ranging from approximately 0.2-2kb which is an appropriate size for a hybridization probe.

Figure 4: 3D-FISH on HeLa cells using a 10kb plasmid as probe (red). Maximum intensity projection of 8 optical sections of a confocal image stack are shown, green represents the DNA counterstain. Click here for a larger view.

Figure 5: FISH on a paraffin tissue section of human skeletal muscles. Maximum intensity projection of a confocal image stack showing nuclei counterstained with TO-PRO-3 (red) and centromeres of all chromosomes. Centromeric regions are visualized by a pancentromeric probe directly labeled with FITC-dUTP by nick-translation. Click here for a larger view.

Figure 6: FISH on a paraffin tissue section of human smooth muscles. Maximum intensity projection of 42 optical sections (c.a. 12.5µm) of a confocal image stack are shown. Nuclei are counterstained with DAPI (blue) centromeres of four chromosomes are visualized by different fluorochromes. Probes for centromeres were alphoid chromosome-specific sequences labeled directly with fluorochromes by nick-translation. Click here for a larger view.

Figure 7: FISH on vibratome sections of mouse retina. Maximum intensity projection of 7 optical sections (ca. 2µm) of a confocal image stack showing nuclei of neuronal cells counterstained with DAPI (blue) with BAC-probes signals targeting four genes, three in green and one in red colours. Since nuclei representing this tissue type extend over approximately 5-7µm in z-diameter, only a fraction of genes is shown in most of the nuclei. Click here for a larger view.

Figure 8: Glass-chamber for hybridization

Figure 9: 6 colour 3D-FISH on nuclei of human fibroblasts. Maximum intensity projection of a confocal image stack with 6 colour channels are shown as original images (upper row) and after linear colour unmixing (bottom row) using the software of Leica SP2. The FITC channel delineates the territories of chromosome 12, Alexa514 of chromosomes 11 and TAMRA the territories of chromosomes 17, 19 and 20. Texas Red delineates a BAC contig of chromosome 11 and Cy5 a BAC pool covering different regions of chromosome 12. White arrows point at the image regions generated due to "leakage" of some fluorochromes to the neighboring channels, e.g. Alexa514 to the FITC channel (and vice versa), orTAMRA to the Texas Red channel. Click here for a larger view.