Preparation of Human Chromosomes for High Resolution Scanning Electron Microscopy
P. O. KÄTTSTRÖM, B. Ove NILSSON
1992
Archives of histology and cytology
The addition of ethidium bromide during the last 2.5-3h of lymphocyte culturing restricted chromosome contraction and preserved the banding structure in scanning electron microscopy. Treatment of the chromosomes with trypsin and use of impregnation with osmium tetroxide and thiocarbohydrazide resulted in a structural preservation of high resolution quality. A method for scanning electron microscopy (SEM) of metaphase chromosomes from human lymphocytes was designed by HARRISON et al. (1985) .
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... ir standard technique for the preparation of chromosomes included incubation of the lymphocytes with colcemid for the collection of metaphases. Usually, however, the chromosomes contract when held in metaphase by colchicine, thus making many bands confluent. Since high resolution banding techniques, which give elongated chromosomes with many distinguishable bands, have been developed for light microscopy (ZABEL et al., 1983; IKEUCHI, 1984) , one aim of this work was to investigate whether a similar technique could be usf ul for SEM. The fine surface structure of chromosomes can be examined by SEM if the metal coating of the preparations is replaced by an impregnation with osmium tetroxide and tiocarbohydrazide (IP and FISCHMAN, 1979; HARRISON et al., 1985) . However, the attainning of high resolution imaging also requires a short trypsin digestion (SEABRIGHT, 1971) , the duration of which has to be found emperically. Therefore, another of our aims was to study the effect of different durations of the trypsinization. MATERIALS AND METHODS Peripheral vein blood was sampled from humans in tubes containing heparin and was processed immediately. The erythrocytes were agglutinated for 30 min at room temperature with phytohemagglutinin and then separated by centrifugation. The leucocytes were cultured in RPMI 1640 medium, supplemented with phytohemagglutinin, penicillin, streptomycin, and 20% fetal calf serum at +3TC in humidified air with 5% CO2. A differential replication staining procedure, modified after BENN and PERLE (1986) , was applied after a culture period of 3 days. 5-bromodeoxyuridin (BrdU) was added at a concentration of 100uM/ml medium, and the cells were grown for 16-17 h. Deoxycytidine was also added at the same time to the same concentration to reduce the toxicity of BrdU. The cells were then rinsed and transferred into a prewarmed thymidine-enriched medium consisting of RPMI 1640, supplemented with penicillin, streptomycin, 20% fetal calf serum, and 2ug/ml thymidine, and the cells were harvested by centrifugation 6 1/2h later. Ethidium bromide (7ug/ml medium) was added during the last 2.5-3h of culturing to restrict the chromosome contraction. Fifteen min before harvest, colcemid was added at a concentration of 5ng/ml medium. The cells were then rinsed and placed into a solution of 75mM KCI, where they were left for 15min. After careful washings, the cells were fixed in a freshly made solution of methanol: glacial acetic acid (3:1). After centrifugation, the cells were suspended in a small volume of fixative, with 5u of this suspension placed on an ethanolwashed and polished circular cover glass (D=12mm), which was air dried at about 40% humidity. Before trypsinization, the cover glasses were rinsed with phosphate buffer to remove any residual fixative, treated with 0.1% Triton in buffer for 30sec, and rinsed in 0.9% saline. The trypsinization was made with 0.025% trypsin in 0.9% saline for 5 or 10 53
doi:10.1679/aohc.55.suppl_53
pmid:1290676
fatcat:spibfn3w4fdfvmkcqq7irc6sve