NanoPlex: A Universal Strategy to Multiplex Immunostainings for Fluorescence Microscopy Using Secondary Nanobodies

Fluorescence microscopy techniques rely strongly on revealing targets of interest using affinity binders, such as antibodies, through a process known as immunostaining or immunofluorescence. Immunostaining is typically achieved using a primary antibody against a specific target of interest, which is then detected by a fluorescently labeled secondary antibody that recognizes the primary antibody's species. This procedure, known as indirect immunofluorescence, constrains the detection of multiple targets by requiring primary antibodies from different species. Here, we describe NanoPlex, a methodology that allows multitarget detection in a species-independent fashion. NanoPlex employs secondary single-domain antibodies (sdAbs, or nanobodies) that enable the simultaneous use of multiple primary antibodies originating from the same species. In addition, these secondary sdAbs are engineered to allow for a mild and specific removal of the fluorescence signal after imaging, enabling a new round of immunostaining and imaging. NanoPlex includes secondary sdAbs with different functional groups, which support directed signal erasing upon application of either UV light (OptoPlex), proteolytic enzyme (EnzyPlex), or a reducing agent (ChemiPlex), fulfilling the demands of various experimental setups. This approach, which involves directed signal removal and re-staining of new targets on the same sample, permits the visualization of multiple targets under the same context in virtually any antibody-based application. Here, we present a detailed protocol for performing NanoPlex on cell cultures, including a practical example for 6-plex in ChemiPlex mode. In this practical example, three iterations are performed while employing dual-color confocal microscopy on each cycle. Notes have been included for step modifications required for EnzyPlex and OptoPlex.