(338e) Multiplexed Three-Dimensional Protein Mapping

A comprehensive map of cellular composition is crucial to systematically study the structure and the function of complex mammalian organs. With the technology advances in high throughput single cell RNA sequencing, cells can be precisely characterized into subtypes based on their gene expression profile. Despite the wide application of single cell RNA sequencing, the transcriptomic information is extracted at the cost of losing the spatial context of each cell. To address the problem, multiple spatial transcriptomics strategies have been developed with a combinatorial barcoding system, which enables high throughput mapping of thousands of gene transcripts within only a few rounds of imaging. However, most applications of spatial transcriptomic tools have been limited to tissues from animal models or surgically dissected human clinical samples due to the high requirement of mRNA preservation. Therefore, their performance is compromised in the cases of long-banked human brain samples, which are clinically important for pathological studies.

Proteomic imaging via immunohistochemistry is another powerful tool to map the cellular identity within tissue context. Proteomic imaging relies on the specific binding between antibody and antigen to detect protein expression and post translational modifications that have direct indications of the cellular identity. With a large pool of robust antibodies developed over decades, most types of tissues, including postmortem human brain samples from brain banks, can be labeled and analyzed based on the expression of proteins of interest or pathological markers. However, the throughput of immunohistochemistry is still limited by the spectral overlap between dyes and the cross-reactivity of antibodies imposed by host animals.

Here, we introduce a highly scalable, multiplexed immunostaining method that enables the extraction of cellular signature based on the simultaneous reading of many proteins. We demonstrated that proteins can be analyzed in a multiplexed and high throughput manner in postmortem human brain tissue sections.