Overcoming Protein A-driven Nonspecific Antibody Staining of <i>S. aureus</i> in Immunofluorescence Microscopy

Abstract The ability to detect host cell factors during Staphylococcus aureus infection in vitro by immunofluorescence microscopy is severely hampered by staphylococcal protein A (SpA), a cell wall-anchored protein that binds the fragment crystallizable (Fc) region of immunoglobulins. This interaction generates strong nonspecific fluorescent signals on the bacterial surface, complicating data interpretation and limiting the accuracy of quantitative image analysis. Several measures have been put forward to overcome this obstacle, most importantly the pre-incubation with an anti-SpA antibody (αSpA) and the use of human serum (HS) as blocking agent and antibody diluent. To highlight this feature to general fluorescence microscopy users, we here systematically evaluated these two strategies. Using S. aureus coated on coverslips and S. aureus -infected A549 cells, we highlight the efficiencies of both approaches to markedly reduce nonspecific fluorescence, with HS treatment yielding the most profound suppression. Notably, HS, containing high levels of human immunoglobulins, offered a robust, cost-effective and broadly applicable solution for minimizing SpA-driven artifacts, thereby improving immunofluorescence microscopy in S. aureus infection models in vitro .