Figure 1

Non-cross- and cross-labelling in indirect immunofluorescence by secondary antibodies. Shown is a typical sequential labelling procedure used for indirect immunofluorescence (IMF) of three target antigens to demonstrate the resulting labelling in two scenarios. (A) Regular state: three different primary antibodies (ABs) origin from rabbit, mouse and goat bind to antigens 1, 2 and 3, respectively. For IMF detection, fluorophore-tagged secondary ABs (“donkey-anti-rabbit Alexa488”, “donkey-anti-mouse Alexa546” and “chicken-anti-goat Alexa594”), which are in origin different from that of targeted primary AB, will not show any cross-labelling. (B) Undesirable cross-labelling: primary ABs for antigens 1, 3 and 4 are originated from rabbit and goat. In this scenario, conjugation of secondary AB for IMF will lead to cross-labelling of rabbit primary AB for antigen 1 with “donkey-anti-rabbit Alexa546” leading to false-positive attribution of “donkey-anti-rabbit Alexa546” AB on antigen 1, bearing the risk of misinterpretation of results such as (co-)localization, spatial distribution, or even molecule-molecule interaction. For easy illustration purposes, a stoichiometry of 1:1 between primary and secondary AB is shown here, however in general, there are on an average more than one fluorophore attached to secondary AB. Similarly, depending on the available binding sites, the number of secondary AB bound to primary AB could vary between 2 to 5.