Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses.

Antibody-based imaging techniques rely on the availability of high-quality reagents whose performance is evaluated for the specific application. Because commercial antibodies are validated for a limited number of uses, in many cases individual laboratories have to employ extensive in-house antibody testing. Here we present a novel strategy to efficiently identify candidate antibodies for array tomography (AT) by introducing an application-specific proxy screening step. AT is a serial section volume microscopy technique that enables quantitative analysis of the cellular proteome in a highly dimensional manner. To identify suitable antibodies for AT-based analysis of synapses in mammalian brain samples, we introduce a heterologous cell-based assay designed to simulate unique features of AT such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included as part of the initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of antibodies for use in AT will further expand the use of this powerful imaging technique.