A rapid method of fluorescent in situ hybridization (FISH) to paraffin-embedded tissues for sexing human embryos and fetuses: Optimal conditions of fixation and proteinase K treatment

Nakamura, N.; Mori, C.; Narita, M.; Uwabe, C.; Shiota, K.

Acta Histochemica et Cytochemica 27(1): 81-87


ISSN/ISBN: 0044-5991
DOI: 10.1267/ahc.27.81
Accession: 008069988

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We describe a rapid fluorescent in situ hybridization (FISH) procedure with X and Y chromosome-specific DNA probes for sexing human embryos and fetuses using paraffin-embedded tissues. The effects of various fixatives and pretreatment conditions on FISH efficiency were examined in different embryonic and fetal tissues. Fluorescent hybridization signals were identified in tissues which had been fixed in 4% paraformaldehyde (PFA) or 10% formalin but not in Bouin-fixed tissues. The preservation in 4% PFA at 4 degree C yielded better FISH results than that in 10% formalin at room temperature. Another critical factor for visualization of fluorescent hybridization signals in paraffin sections was the duration of proteinase K digestion. Optimal digestion visualized hybridization signals more clearly probably because it increased the accessibility of DNA probes into nuclear DNA in paraffin sections. We have succeeded in shortening the hybridization time significantly and eliminating an immunocytochemical step for amplifying hybridization signals, and it has become possible to sex human conceptuses in paraffin sections within 5 hr. This FISH technique should be useful to sex fixed human embryos and fetuses retrospectively and to diagnose sex chromosome aneuploidies using fixed tissues.