Intracellular markers

An increased level of complexity will be encountered when developing protoc ols for intracellular markers. Protocols for surface markers have been succ essfully standardized, however it is understood that no single method is ap propriate for all intracellular staining. A systematic approach should be f ollowed, including knowledge of antigen location and functional state, sele ction of cell fixative and cell permeabilizer, antibody specificity and cla ss/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 si ngle technique to stain all intracellular antigens, the authors present a l ogical approach to follow when developing a staining protocol. (C) 2000 Els evier Science B.V. All rights reserved.