Many commercial ABs are sold in solutions containing azide, which can be toxic to cells even at low concentrations. It is usually crucial to buy azide-free AB solutions, or to remove the azide e.g. by dialysis.
Control experiments should be performed to test if the AB binds the correct molecules under the used conditions. Experiments with cells that do or do not express the epitope to be detected, as well as with isotype control ABs, allow testing if the AB binds to all and only the correct cells. In addition, cells can be analyzed after “in culture live staining” by subsequently co-staining them with ABs against the same molecule labeled with a different fluorophore and analysis by e.g. FACS. When using an AB against a different epitope of the same antigen, all and only cells stained by in culture AB binding should also be bound by the second AB. Alternatively, when using an AB against the same epitope (e.g. the same AB clone labeled in a different color), binding of the second AB should be decreased or inhibited by the first AB already bound during culture (Figure 1).
It is important to keep in mind that some ABs can inhibit or activate functions of their target molecules8 and could influence the behavior of the cells to be stained. Control cultures without ABs should always be observed in parallel to exclude possible effects of the ABs on the analyzed biological system.
Cells can take up bound ABs via receptor-mediated endocytosis, possibly leading to long lasting labeling of endocytotic vesicles. Although this can improve detection of cells expressing an antigen by continuous increase of an otherwise low signal, it can also prevent the detection of surface molecule down-regulation. In this case, labeling of ABs with pH sensitive dyes like FITC will lead to loss of fluorescence upon internalization into vesicles with low pH and allow visualization of only freshly bound epitopes on the cells’ surface.