Fast multicolor primed in situ protocol for chromosome identification in isolated cells may be used for human oocytes and polar bodies  Franck Pellestor, Ph.D., Tal Anahory, M.D., Brigitte Andréo, M.Sc., Gilles Régnier-Vigouroux, M.D., Jean Pierre Soulié, M.D., Magalie Baudouin, M.Sc., Jacques Demaille, M.D.  Fertility and Sterility  Volume 81, Issue 2, Pages 408-415 (February 2004) DOI: 10.1016/j.fertnstert.2003.08.014

FIGURE 1 Principles of the fast multicolor PRINS reaction (modified from Yan et al. [9]). Four sequential PRINS reactions, using specific centromeric primers for four chromosomes and distinct fluorochrome-dUTPs (FITC-11-dUTP, TRITC-6-dUTP, Cascade Blue-7-dUTP) were performed on a chromosome preparation, without intermediate 3′-end blocking steps. Thus, the in situ mixing of the colors led to the final generation of four distinct colored spots (green, red, yellow, and blue), allowing the specific identification of each targeted chromosome. In the present illustration, chromosome 1 was successively labeled by the in situ incorporation of FITC, TRITC, FITC, and Cascade Blue, leading to a final yellow color. Chromosome 9 was in situ labeled by FITC, TRITC, and Cascade Blue, leading to a red color. Chromosome 16 was labeled by the incorporation of FITC and Cascade Blue, resulting in a green color. Finally, chromosome 18 only incorporated Cascade Blue dUTP, giving a blue color. Open circles, dNTP; green circles, FITC-dUTP; red circles, TRITC-dUTP; and blue circles, Cascade Blue–dUTP. Pellestor. Ultrarapid PRINS on human cycles.Fertil Steril 2004. Fertility and Sterility 2004 81, 408-415DOI: (10.1016/j.fertnstert.2003.08.014)

FIGURE 2 Examples of labeling using three- or four-color PRINS protocol. (A) Triple-color PRINS labeling of chromosome 1 (yellow), chromosome 7 (green), and chromosome 9 (red) on metaphase and interphase nuclei, using the labeling order FITC-TRITC-FITC. (B) Four-color PRINS labeling of chromosomes 1 (red), 9 (yellow), 16 (blue), and 18 (green) using the labeling combination FITC-TRITC-FITC–Cascade Blue. (D) Triple-color labeling of a human oocyte. Chromosome 1 is labeled in yellow, chromosome 7 in green, and chromosome 16 in red. (D) Labeling of the corresponding first polar body displaying the same labeling as in panel C. (E) Triple labeling of both oocyte and first polar body using the combination of chromosome 1 (yellow), chromosome 9 (red), and chromosome 18 (green). The yellow labeling of chromosome 1 is obtained only in oocyte chromosomes. (F) Four-color labeling of human oocyte 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. (G) Corresponding first polar body and PCC (arrowhead) displaying the identical labeling of chromosomes 1, 7, 9, and 16. (H) Four-color labeling of an oocyte with chromosome 1 in red, chromosome 9 in yellow, chromosome 16 in green, and chromosome 18 in blue. This oocyte displays an additional centromeric signal for chromosome 16 (arrow). This additional signal indicated the presence of a supernumerary chromatid (+cht16) on this metaphase II chromosome spread, and consequently the previous occurrence of the premature separation of the two chromatids of chromosome 16 through meiosis I. (I) Combined PRINS-FISH labeling of an oocyte II chromosome spread. Chromosomes 1, 7, and 9 were labeled by PRINS, in red, green, and yellow, respectively. Chromosomes 18 (in bright yellow), 20 (in orange), and X (in blue) were subsequently labeled by a multicolor FISH reaction. Pellestor. Ultrarapid PRINS on human cycles.Fertil Steril 2004. Fertility and Sterility 2004 81, 408-415DOI: (10.1016/j.fertnstert.2003.08.014)