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

Area of Science:

• Immunohistochemistry
• Microscopy techniques
• Proteomics

Background:

• Antibody (Ab)-based imaging requires application-specific reagents.
• Commercial antibodies often lack validation for diverse research applications, necessitating extensive in-house testing.
• Array tomography (AT) is a microscopy technique for high-dimensional quantitative analysis of the cellular proteome.

Purpose of the Study:

• To introduce a novel, application-specific proxy screening step for identifying candidate antibodies for array tomography (AT).
• To develop a heterologous cell-based assay simulating AT conditions to pre-validate antibodies for synaptic analysis in mammalian brain.
• To simplify antibody screening and enhance the utility of AT for cellular proteome research.

Main Methods:

• A heterologous cell-based assay was designed to mimic AT fixation and resin embedding conditions.
• This assay was integrated into an initial screening strategy for generating monoclonal antibodies for AT.
• A database of AT-validated antibodies, with a neuroscience focus, was compiled.

Main Results:

• The proxy screening step demonstrated high predictive value for identifying antibodies suitable for AT.
• The developed assay efficiently identifies candidate antibodies, reducing the need for extensive in-house validation.
• Validated antibodies showed high success rates for postembedding applications, including immunogold electron microscopy.

Conclusions:

• The novel screening approach significantly simplifies antibody selection for AT.
• The developed AT-compatible antibody toolbox is expanding, enhancing the capabilities of this imaging technique.
• This strategy improves the efficiency and reliability of antibody validation for advanced microscopy applications.