  Antibodies are widely used in life-sciences and medical therapy. Yet, broadly
applicable methods are missing to determine, in the biological system of
choice, antibody specificity and its quantitative contribution to e.g.
immunofluorescence stainings. Thereby, antibody-based data often needs to be
seen with caution. Here, we present a simple-to-use approach to characterize
and quantify antibody binding properties directly in the system of choice. We
determine an epitope accessibility distribution in the system of interest based
on a computational analysis of antibody-dilution immunofluorescence stainings.
This allows the selection of specific antibodies, the choice of a dilution to
maximize signal-specificity, and an improvement of signal quantification. It
further expands the scope of antibody-based imaging to detect changes of the
subcellular nano-environment and allows for antibody multiplexing.
