Indirect Immunofluorescent Labeling of Fixed Cells

Abstract

One of the major advantages of flow cytometry is the simultaneous evaluation of multiple markers, especially surface markers (1). The detection of intracellular proteins is less well developed, in large part because antibodies can bind nonspecifically to dying cells and dead cell components, which leads to considerable biological noise in the fluorescence detectors. There is also noise caused by the intra-and/or intermolecular ionic interactions during the process of fixation, which reduces the ability to detect the desired protein(s) both intracellularly and extracellularly. This is a double edged sword for labeling cells. It is very important to start the staining procedure with a viable cell suspension, If the starting material is viable, at least one of the two problems associated with cell fixation and staining is remedied. The fixation protocols are varied not only for their uses of crosslinking agents, permeabilization agents, and/or precipitating agents, but also for time and temperature. This chapter includes protocols for surface staining followed by fixation/ permeabilization for DNA staining, fixation/permeabilization followed by surface staining, and fixation/permeabilization followed by intracellular staining.