In direct immunofluorescence staining, cells are incubated with an antibody directly conjugated to a fluorochrome (e.g. FITC). This has the advantage of requiring only one antibody incubation step and eliminates the possibility of non-specific binding from a secondary antibody.
This approach is particularly useful for intracellular staining, where large antibody-fluorochrome complexes including secondary antibodies can become trapped causing non-specific binding, or fail to enter the cell preventing primary antibody detection.
In indirect staining, the primary antibody is not fluorochrome-labeled, but is detected by a fluorochrome-labeled secondary antibody. This second reagent may be an antibody with specificity for the first antibody. Alternatively, the avidin-biotin system can be used, whereby an antibody is conjugated to biotin and detected with fluorochrome-labeled avidin.
With the wide range of conjugated antibodies now available, this method means that unconjugated primary antibodies raised against many different targets can be used in conjunction with a labeled secondary antibody for FACS analysis. This widens the choice of target proteins for the researcher.
taining of intracellular antigens for flow cytometry protocols depends on various fixation and permeabilization methods to allow access of antibodies to internal cellular proteins. A successful staining procedure in all cases is dependent on optimization of experimental conditions through titering of antibodies, use of appropriate controls to set up the flow cytometer correctly and optimized fixation and permeabilization procedures.
Detection of secreted proteins is difficult as the protein will be released from the cell before detection, or may degrade rapidly. A Golgi block such as Brefeldin A can be used to inhibit secretion of expressed proteins, retaining them in the Golgi apparatus. The intracellular staining method can then be used for detection of the target protein.