Detection and Microscopy

1. Drain the excess washing solution off the slide.

2. Mount the slide in Vectashield antifade solution containing either DAPI (0.3 pL/mL) or a mix of propidium iodide (0.3 pL/mL) and DAPI (0.3 pL/mL).

3. Cover with a 22 x 40-mm cover slip and seal the cover slip with rubber cement.

4. Examine the slide under the epifluorescence microscope, preferentially using first the triple or double band-pass filter, and confirming the coloration of the fluorescent spot with single band-pass filters. Figure 1 shows an example of PRINS labeling performed on human oocyte chromosomes.

Fig. 1. Four-color PRINS labeling of human oocyte chromosomes using the labeling order FITC/TRITC/FITC/Cascade Blue. Chromosome 1 is labeled in yellow, chromosome 7 in green, chromosome 9 in red, and chromosome 16 in blue. (Please see color insert following p. 48.)

4. Notes

1. Make sure that the pipet is free of oil inside and outside. Excess oil must be removed from the pipet to avoid the formation of an oil layer on the solution surface. The presence of oil on the slide interferes with the proper fixation of the cell. Two watch dishes can be used to prevent the transfer of oil to the slide.

2. The fixation is the key to the success of the entire technique. Fixation removes the cytoplasm material and makes the nuclear DNA accessible to subsequent labeling procedure. Relative humidity is essential in the area where cell fixation takes place. A relative humidity of 40 to 50% must be maintained. It is also important that standard fluorescent lighting is available above the area where fixation will perform so that egg can be observed by the reflection of the light and solution spreading on the slide can be observed.

3. At this point, a rainbow formation visible under the standard ceiling fluorescent lighting will begin moving in toward the center of the slide where the cell is.

Allow the rainbow to come within 0.5 cm on either side of where the egg is before dropping a new drop of fixative.

4. Blastomere fixation was initially performed using classical Tarkowsky technique using methanol/acetic acid as a fixative agent. After the publication of the HCl-Tween-20 technique (7), some laboratories began using this alternative procedure, which allows to monitor the lysis of the cell and limits scattering of nuclear material.

5. First, the cell membrane is ruptured and the nucleus is separated from the bulk of the cytoplasm. The shape of the blastomere may distort and at this stage, the nucleus should be visible. Keep watching the nucleus. The nucleus can be obscured by the addition of too much spreading solution and lost from inconsistent observation. If at any point you lose sight of it, stop and let the slide dry.

6. Pepsine is used to remove excess cytoplasmic proteins on the slide. The removal of cytoplasmic remnants is critical. If the pepsin treatment is not sufficient, the remaining cytoplasmic proteins will prevent the binding of probes or primers. On the other hand, overexposure to pepsin may lead to the degradation of targeted DNA. Be careful because the pepsine activity can vary from batch to batch.

7. For the three-color PRINS reactions, the combination order FITC/TRITC/FITC give the best results, with well distinct red, green, and yellow spots. When using the reversed labeling combination TRITC/FITC/TRITC, no pure yellow color, but a mixed orange color is obtained for the first labeled chromosome. Indeed, a mean ratio of green to red color of 70%:30% must be respected to obtain well-defined green, red, and yellow signals. In the four-color procedure, the addition of the blue label does not affect the final coloring of the three previously labeled targets. The Cascade Blue dye provides a color which contrasts well with the longer-wavelength green and red fluorophores.

Acknowledgment

The experiments performed for elaborating this protocol were supported by a European INTAS project no. 03-51-4060.

References

1. Egozcue, J., Blanco, J., and Vidal, F. (1997) Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH). Hum. Reprod. Update. 3, 441-452.

2. Harper, J. C., and Delhanty, J. D. (1996) detection of chromosome abnormalities in human preimplantation embryos using FISH. J. Assist. Reprod. Genet. 13, 137-139.

3. Pellestor, F., Imbert, I., and Andreo, B. (2002) Rapid chromosome detection by PRINS in human sperm. Am. J. Hum. Med. 107, 109-114.

4. Koch, J. E., Kolvraa, S., Petersen, K. B., Gregersen, N., and Bolund, L. (1989) Oligonucleotide-priming methods for the chromosome-specific labeling of alpha-satellite DNA in situ. Chromosoma 98, 259-265.

5. Coignet, L., Girardet, A., Andreo, B., Charlieu, J.-P., and Pellestor, F. (1996) Double and triple in situ chromosomal labeling of human spermatozoa by PRINS. Cytogenet. Cell. Genet. 73, 300-303.

6. Yan, J., Bronsard, M., and Drouin, R. (2001) Creating a new color by omission of 3 end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique. Chromosoma 109, 565-570.

7. Coonen, E., Dumoulin, J. C., Ramaekers, F. C., and Hopman, A. H. (1994) Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization. Hum. Reprod. 9, 533-537.

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