Observation and Image Acquisition

1. Classical acquisitions are made using a Zeiss epifluorescent microscope equipped with tri-CCD camera and Vysis computer software (Smart capture 2). An example of simultaneous labeling obtained is illustrated in Fig. 2 with the following specific labeling: chromosome centromeres labeled in green by PRINS, achromatic spindle in red by IIF of the P tubulin, and the whole DNA in blue using DAPI as counterstains.

2. Confocal acquisitions: Refined images are captured using a Zeiss LMS 510 laser scanning confocal microscope, either with Planapochromat X63/NA 1.4 or x100/ NA1.4 objectives. The three laser excitations are used: 488 nm, 543 nm, and 633 nm, for FITC, TRITC, and TOTO 3, respectively (see Table 2). Serial optical sections of 0.7 mm (image collected at 0.5-mm intervals) along the z-axis of the cell are collected sequentially for each marker and overlaid to obtain a three-dimensional reconstruction. Figure 3 provides an example of such labeling obtained on endomitotic megacaryocytes.

4. Notes

1. The cells lines and the culture conditions described here correspond to the set of problems that we had to solve and to our original publications (3,7), but the methodology can be easily transposed to other types of cells.

2. Solutions 1X PBS, 2X SSC, and 4X SSC-Tween, prepared from commercial concentrated solutions, can be stored several weeks at 4°C.

3. The fixative solution is always prepared extemporaneously. Notice that for a better preservation of the antigenic sites, the percent of acetic acid is reduced in

Fig. 2. Simultaneous PRINS (human chromosome 1 centromeres) and IIF (achromatic spindle) on diploid cells (KG1a): Primer 1c and fluorescein-16-dUTP were used for PRINS and the targeted centromeres are labeled in green. Microtubules were stained in red by indirect immunofluorescence with an anti-P tubulin antibody, followed by incubation with TRITC anti-mouse F(ab')2 fragment. The whole DNA is counter-stained in blue by DAPI. The picture acquisition was made using a Zeiss epifluorescent microscope using a tri-CCD camera and Vysis computer software (Smart capture 2). We observed two green dots, corresponding to the centomeres of the two targeted chromosomes 1, at prophase (A) and metaphase (B). Four dots, equally distributed on both sides of the cytokinetic apparatus, were present when sister-chromatids and centromeres separated from the beginning of anaphase to the onset of cytokinesis, two on each side of the equator (C). (Color version of this figure available in ebook.)

Fig. 2. Simultaneous PRINS (human chromosome 1 centromeres) and IIF (achromatic spindle) on diploid cells (KG1a): Primer 1c and fluorescein-16-dUTP were used for PRINS and the targeted centromeres are labeled in green. Microtubules were stained in red by indirect immunofluorescence with an anti-P tubulin antibody, followed by incubation with TRITC anti-mouse F(ab')2 fragment. The whole DNA is counter-stained in blue by DAPI. The picture acquisition was made using a Zeiss epifluorescent microscope using a tri-CCD camera and Vysis computer software (Smart capture 2). We observed two green dots, corresponding to the centomeres of the two targeted chromosomes 1, at prophase (A) and metaphase (B). Four dots, equally distributed on both sides of the cytokinetic apparatus, were present when sister-chromatids and centromeres separated from the beginning of anaphase to the onset of cytokinesis, two on each side of the equator (C). (Color version of this figure available in ebook.)

Fig. 3. Asymmetrical repartition of chromosome 1 on multipolar spindle of endomitotic megakaryocytes. Double staining obtained respectively by PRINS (green) and IIF (red) procedure in endomitosis 4n to 8n (A), 8n to 16n (B), and 16n to 32n (C). The blue whole DNA counterstain, evidenced by TOTO3, was not integrated on this picture. Centromeres of chromosomes 1 are asymmetrically distributed among the different spindle-poles because some of them are not associated with green dot. Images were captured using a Zeiss LMS 510 laser scanning confocal microscope Zeiss. (Color version of this figure available in ebook.)

Fig. 3. Asymmetrical repartition of chromosome 1 on multipolar spindle of endomitotic megakaryocytes. Double staining obtained respectively by PRINS (green) and IIF (red) procedure in endomitosis 4n to 8n (A), 8n to 16n (B), and 16n to 32n (C). The blue whole DNA counterstain, evidenced by TOTO3, was not integrated on this picture. Centromeres of chromosomes 1 are asymmetrically distributed among the different spindle-poles because some of them are not associated with green dot. Images were captured using a Zeiss LMS 510 laser scanning confocal microscope Zeiss. (Color version of this figure available in ebook.)

comparison with the conventional cytogenetic fixative (25%), and the duration of the fixation itself is as short as possible.

4. For a better conservation, we store the formamide and, particularly, the opened flasks at 4°C in the dark. The 70% formamide solution used for denaturation also can be conserved at 4°C in the dark, preventing evaporation. However, remove the solution after four reaction runs or after a month.

5. We experimented using a different sequencing of the method, but we observed that the tubulin epitope was far better preserved when immunostaining was performed after DNA labeling.

6. If our cell spreading system cells don't require cover slips, it is advantageous to use slides because cover slips can be easily broken.

7. The PRINS reaction is sensitive to DNA breaks. It is important that the slides that will are frozen be perfectly dried. Opening the boxes after a 37°C incubation prevents the formation of condensation on the slides. In the same way, after closing the boxes, we let them sit for 30 min at room temperature before putting them at -20°C so that the desiccator operates well on the atmosphere of the box.

8. First prepare an 10% triton solution in distillated water.

9. It is important that pH is 7.0-7.2. In the same way, it is important that the denatur-ation temperature is truly 71°C and the duration of denaturation is respected. Consequently, we operate slide by slide. The first denatured slides can delay in the last ethanol bath.

10. If you are not ready to perform the PRINS reaction, you can delay it by keeping the slides in the last ethanol bath; this tactic also can be used for the immunofluorescence procedure.

11. All the PRINS reagents are stored at -20°C. We always add the Taq polymerase to the mix just before starting the PRINS reaction.

12. For a better efficiency, do not drive more than four slides together. If cell preparations are on slides, of course, put the slides on the block and, after putting the mix, protect with cover slips and seal with rubber cement. Protect this preparation from the light.

13. A high temperature (72°C during elongation) is used for the elongation step, implying that the studied epitope must be heat resistant. No problems were discovered in our own experiments.

14. 25 pL of antibody solution should be placed between cover slip and slide. Rubber cement is not required, but incubate at 37°C in a moist chamber. Incubations also could be made at room temperature. Don't forget to protect the preparation from light.

15. Put in suspension the original lyophilized DAPI in 1 mL of distillated water. Prepare a 1/100 solution. For each 200 pL of Vectashield, add 1 pL of this solution. We store all reagents at -20°C.

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