Van Furth R, Van Zwet T L
Department of Infectious Diseases, University Hospital, Leiden, The Netherlands.
J Immunol Methods. 1988 Apr 6;108(1-2):45-51. doi: 10.1016/0022-1759(88)90401-2.
5-Bromo-2-deoxyuridine (BrdUrd) is a thymidine analogue which can be used to detect DNA synthesis in cells. The present paper describes a method for the fixation and DNA denaturation of cells that affects neither the morphology of the cells not their adhesion to a glass surface. The cells were fixed in absolute ethanol and glacial acetic acid followed by gradual rehydration. Denaturation of DNA was achieved by very brief exposure of the cells to low pH, followed by heating to high temperature and rapid cooling. Incorporation of BrdUrd was assessed from the binding of monoclonal antibody to BrdUrd in single-stranded DNA as detected with an immunocytochemical method. This method makes it possible to demonstrate BrdUrd labeling of individual, morphologically identifiable cells. The results obtained with this method correspond well with the autoradiographic data on [3H]thymidine incorporation.
5-溴-2'-脱氧尿苷（BrdUrd）是一种胸腺嘧啶类似物，可用于检测细胞中的DNA合成。本文描述了一种细胞固定和DNA变性方法，该方法既不影响细胞形态，也不影响其与玻璃表面的黏附。细胞先用无水乙醇和冰醋酸固定，然后逐步复水。通过将细胞短暂暴露于低pH值，随后加热至高温并快速冷却来实现DNA变性。用免疫细胞化学方法检测单链DNA中BrdUrd与单克隆抗体的结合，以此评估BrdUrd的掺入情况。该方法能够显示形态可识别的单个细胞的BrdUrd标记。用此方法获得的结果与关于[3H]胸腺嘧啶掺入的放射自显影数据非常吻合。
