Phenomenon of selective staining of pericentromeric heterochromatin regions in chromosomes from spontaneously dividing cells by the acridine orange fluorochrome

Abstract

369 † Various methods are used for detection of hetero chromatin regions (HRs) of chromosomes. The CBG method is the most versatile; it allows the staining of centromeric and pericentromeric HR of all chromo somes, as well as a HR in the long arm of the human Y chromosome. Different variants of C banding using Giemsa staining make it possible to image only active centromeric regions (Cd staining) or pericentromeric HRs of individual chromosomes (G11 staining) [1,2]. The fluorochromes D287/170, DAPI, and Hoechst 33258 in combination with contrasting agents allows the staining of only polymorphic HRs of chromo somes 1, 9, 15, 16, 22, and Y [3].The acridine orange (AO) fluorochrome has been used in human cytogenetics for a long time. AO is metachromatic due to the characteristics of its inter action with nucleic acids (NAs), proteins, and other biopolymers [4,5]. In a solution, AO may exist in either the monomeric form, which intercalates into double stranded NA and stains it green, or in the dimeric form, which electrostatically binds with sin gle stranded NA and stains it red. AO is known to stain chromosomes yellow-green, relatively homoge neously, in non pretreated specimens. For AO band ing, including selective staining of HRs, the sample or chromosome slide should be pretreated to denature or † Deceased. decondense chromatin (e.g., the RFA and RBA meth ods) [1,[5][6][7].Earlier, we found bright red fluorescence of the 1qh, 9qh, 16qh, and Yqh regions after AO staining of chorionic villus slides prepared by the direct method and not subjected to any additional treatment [8]. This unexpected finding stimulated our studies on the char acteristics of AO staining of specimens prepared by different methods from cells of different origins. MATERIALS AND METHODS Human metaphase chromosome preparations from samples of the chorion, placenta, and embryonic organs.Samples of chorionic villi (41 samples) and embryonic organs (the liver, kidney, brain, intestine, lung, and eye) of ten human embryos were obtained after thera peutic abortions for medical or social reasons at a ges tational age of 5-12 weeks, as well as samples of 12 placentas performed by Caesarian section at a ges tational age of 36-40 weeks.The "quick direct" method [9]. Chorionic villi or fragments of organs were put into a hypotonic solution containing colchicine at a final concentration of 0.8 µg/mL. The hypotonic solution was 0.9% sodium citrate. For three samples in parallel experiments, 1% KCl was used. Then, the samples were prefixed at Abstract-A novel phenomenon of unusual selective acridine orange (AO) staining of pericentromeric het erochromatin regions (HRs) in chromosomal preparations from tissue with known spontaneous mitotic activity (chorionic villi, placenta, embryonic tissues, bone marrow, and testes), as well as embryonic stem cells, is described. Staining with 0.01% AO in a citric-phosphate (pH 5.5) or sodium phosphate (pH 7.0) buffer solution allows the HRs of human chromosomes (1q12, 9q12, 13p11.2, 14p11.2, 15p11.2, 16q11.2, 21p11.2, 22...

Prevalence of chromosomal alterations in first-trimester spontaneous pregnancy loss

“…Demecolcine (Sigma-Aldrich) was added 4 h before chromosome harvesting, and the samples were processed using standard techniques of hypotonic treatment with 0.55% sodium citrate and cell fixation with a 1:3 mixture of acetic acid:methanol. In some cases, direct preparations of the chorionic villi were used 69 . Slide preparations and GTG banding were performed by standard protocols in accordance with guidelines 70 .…”