Resources for Three-Dimensional Proteomic-Based Cellular and Tissue Structures

mAb3D Atlas

Our aim is to create a three-dimensional whole-brain reference atlas of protein expression in the adult mouse brain. This atlas will reveal underlying brain architecture and provide a platform for scientists to examine cellular and organ level changes in biomedical research and diagnosis.

Toward this end, we are actively building a reference list of monoclonal antibodies compatible with immunohistochemistry and the iDISCO family of whole-mount methods. We use these antibodies to spatially profile the expression of diverse proteins at cellular resolution in the mouse brain in both 2D and 3D. To facilitate translational insights across species, we further characterize many of these antibodies on rat, marmoset, and human brain tissue. Concurrently, we develop protocols for improved tissue clearing and whole-mount imaging.

By sharing these resources with the neuroscience community, we hope to expand fellow laboratories' experimental capabilities and to promote reproducible brain proteomic analysis with the validated monoclonal antibody resources and references.

Why monoclonal antibodies?

Why study neural structures in 3D?

Monoclonal antibodies (mAb) provide a reliable, semi-quantitative means of characterizing protein expression and distribution in biological tissues. Produced by hybridoma cell lines (or more recent recombinant techniques), monoclonal antibodies can be generated in vitro, allowing a renewable and ethical means of long-term antibody production. Most importantly, the monoclonal antibody production approach also minimizes lot-to-lot variation in antibody performance.

An additional benefit to monoclonal antibodies is that each clone binds a single epitope on the target protein. Due to this one-to-one binding ratio, relative levels of specific protein expression can be assessed with greater confidence by immunohistochemistry following standard procedures.

Since monoclonal antibodies are a “renewable” and reliable resource, the data we produce and share on this website will be useful to the community for long term reference and cross-comparison.

Though a neuron’s cell body is small, its projections may extend across the entire brain, branching tens to hundreds of times and intertwining with other neurons’ projections along the way. Due to their complex three-dimensional morphology, neuronal structures are hard to profile with traditional section-based histology (where, for example, transverse axons appear as small dots). Along the same lines, brain protein distributions and functions are likely not to be refined to the original cell body regions, but exhibit rather complex three-dimensional patterns and attributes.

Imaging the intact brain in three-dimensions provides a more accurate and complete characterization of the structures which compose the brain’s functional circuitry. Through this method, we can also more effectively assess changes in proteomic distribution that occur across the brain due to genetic disorders or disease.

While our interest and focus are on the nervous system, our protocols and resources could be adapted to other tissues and organs across broad animal species to appreciate organ/system level molecular patterns and cellular interactions.

Production Overview

mAb3D Database

Production

We have to-date screened over 300 mAbs to assess their staining pattern and strength via immunohistochemistry on 100μm-thick sagittal mouse brain sections. Tissue sections are prepared and labeled using collection, delipidation, and thick-section IHC methods designed to recapitulate those used for whole-mount staining and imaging. Results provide an indication of each antibody’s suitability for use in whole-mount. Our antibody-screening pipeline has been refined to balance both robustness and simplicity to produce reliable and optimized results. Details about our pipeline methodology can be found below.

Click to Enlarge

Resource Overview

Protocol References

Suggested reagents for sample collection, delipidation, and thick-section IHC protocols.

Validated Monoclonal Antibody Database

Visit our database of monoclonal antibodies that have been validated in human, marmoset, and mouse species.

idisco.info

For additional whole-mount immunolabeling and tissue clearing protocols, see idisco.info.

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