Koester S K, Bolton W E
Beckman Coulter, Inc., Advanced Technology, 11800 S.W. 147th Avenue, M/C 22-A01, 33196-2500, Miami, FL, USA.
J Immunol Methods. 2000 Sep 21;243(1-2):99-106. doi: 10.1016/s0022-1759(00)00239-8.
An increased level of complexity will be encountered when developing protocols for intracellular markers. Protocols for surface markers have been successfully standardized, however it is understood that no single method is appropriate for all intracellular staining. A systematic approach should be followed, including knowledge of antigen location and functional state, selection of cell fixative and cell permeabilizer, antibody specificity and class/subclass, fluorochrome, fluorochrome to protein ratio (F:P), and use of adequate controls, including isotype-matched negative controls and positive and negative cell controls. Even though it is impossible to recommend a single technique to stain all intracellular antigens, the authors present a logical approach to follow when developing a staining protocol.
在开发细胞内标志物检测方案时，会遇到更高的复杂性。表面标志物的检测方案已成功实现标准化，然而，人们明白没有一种单一的方法适用于所有细胞内染色。应遵循一种系统的方法，包括了解抗原的位置和功能状态、选择细胞固定剂和细胞通透剂、抗体特异性和类别/亚类、荧光染料、荧光染料与蛋白质的比例（F:P），以及使用适当的对照，包括同型匹配的阴性对照和阳性及阴性细胞对照。尽管不可能推荐一种单一的技术来染色所有细胞内抗原，但作者提出了一种在制定染色方案时应遵循的合理方法。
