Camplejohn R S
Richard Dimbleby Department of Cancer Research, UMDS, St Thomas Hospital, London, U.K.
J Microsc. 1994 Oct;176(Pt 1):1-7. doi: 10.1111/j.1365-2818.1994.tb03493.x.
The aim of this paper is to provide a strategy for measuring intracellular antigens combined with DNA content in cells or nuclei. A series of protocols are included which enable the majority of such antigens to be labelled and further information is provided for cases in which the standard methods prove to be inadequate. The basic principles of cell permeabilization/fixation are described, thus explaining how methods can be divided into three basic categories: (a) alcohol fixation with or without detergent pretreatment; (b) paraformaldehyde fixation followed by permeabilization with alcohol or detergents; (c) permeabilization of unfixed cells. The preparation of nuclear suspensions from paraffin-embedded material is described and the possibilities and problems of staining such suspensions for nuclear antigens are discussed. Examples of results obtained with the detailed protocols are given for staining with antibodies directed against proliferating cell nuclear antigen (PCNA), Ki-67 antigen and Ki-S1 antigen. Details of published studies of a variety of intracellular antigens are given in two tables. The power of multiparametric flow cytometry in the study of cell proliferation, differentiation and response of cells to damage is underlined.
本文旨在提供一种在细胞或细胞核中测量细胞内抗原并结合DNA含量的策略。文中包含一系列方案，可对大多数此类抗原进行标记，并针对标准方法证明不足的情况提供了更多信息。描述了细胞通透/固定的基本原理，从而解释了方法如何分为三大类：(a) 有无去污剂预处理的酒精固定；(b) 多聚甲醛固定后用酒精或去污剂通透；(c) 未固定细胞的通透。描述了从石蜡包埋材料制备核悬液的方法，并讨论了对这种核悬液进行核抗原染色的可能性和问题。给出了针对增殖细胞核抗原(PCNA)、Ki-67抗原和Ki-S1抗原的抗体染色详细方案获得的结果示例。两个表格中给出了各种细胞内抗原已发表研究的详细信息。强调了多参数流式细胞术在细胞增殖、分化及细胞对损伤反应研究中的作用。
