direct Stochastic Optical Reconstruction Microscopy to Determine the Oligomeric State of Proteins on the Plasma Membrane and Their Accessibility for Immunotherapeutic Antibodies.

Super-resolution fluorescence microscopy enables the visualization of protein structures at nanometer resolution, providing insights into receptor organization on the plasma membrane that are essential for the development and optimization of immunotherapies. In this context, monoclonal antibodies are employed, which typically bind only a subset of available membrane receptors, due to steric hindrance or otherwise limited epitope accessibility, to quantify the accessible targets. These accessible targets, rather than the total receptor density, are critical for determining therapeutic efficacy. Here, we present a simplified, robust protocol to quantify antibody-accessible endogenous receptors using monoclonal antibodies directly labeled with fluorescent dyes in combination with total internal reflection fluorescence (TIRF) direct stochastic optical reconstruction microscopy (dSTORM). The method employs optimized labeling and fixation conditions to preserve the native receptor distribution, enabling precise quantification of accessible receptors and their stoichiometry at single-molecule resolution. Omitting secondary antibodies and minimizing fixation-induced artifacts prevents artificial clustering and maintains the physiological binding pattern of therapeutic antibodies. The standardized workflow delivers therapy-relevant information about receptor accessibility and organization underlying therapeutic antibody binding, thereby advancing the mechanistic understanding of immunotherapy resistance and personalized treatment strategies across diverse membrane protein targets. Key features • Influence of fixation conditions on receptor epitope accessibility. • Use of therapeutic antibodies for quantitative estimation of receptor availability relevant to immunotherapy. • Fluorophore localizations provide information on antibody binding events below the optical resolution limit.