# 3D molecular phenotyping of intact brain tissue via high-throughput active immunohistochemistry

> **NIH NIH R44** · LIFECANVAS TECHNOLOGIES, INC. · 2022 · $350,347

## Abstract

Abstract. Molecular phenotyping has led to a growing appreciation of neural cell type diversity and function
thereby transforming our understanding of the brain. Beyond first-order classification of cells as glial or neuronal,
excitatory or inhibitory, it is now recognized that there are dozens of molecularly-defined cell types that differ in
their morphology, connectivity, physiology, and gene & protein expression. Detection of protein in fixed tissues
via immunohistochemistry (IHC) has been a major driver of cell type discovery, with a cell’s precise
microenvironment within tissue (e.g. proximity to vasculature and extracellular deposits) providing essential
physiological context. Such data have yielded a rich picture of how cellular topography varies by brain region
and provides a robust backdrop to assess cell type-specific changes that occur in disease states, such as
profound neurological disorders like Alzheimer’s and Parkinson’s disease. Despite the prevalence of IHC, its
application has remained encumbered by the slow rate at which reagents such as IgG antibodies passively
diffuse into tissue. Due to this bottleneck, tissues have traditionally been thinly sliced (≤ 50 µm) to facilitate
uniform staining of features and make quantitative analyses reliable. CLARITY, iDISCO, and related techniques
that optically-clear intact tissues by removing cell membrane lipids have offered a means to perform whole-brain
IHC, as delipidation grants reagents easier access to deep tissue sites. However, labeling time remains a major
bottleneck, with intact samples requiring weeks to months of incubation for labeling to reach the center. If whole
organs could be labeled more quickly and practically it would provide a powerful tool to perform unbiased
molecular phenotyping in mammalian models of development and neurological disorders. To this end LifeCanvas
developed SmartLabel (SL), the world’s first whole-organ active immunostaining device that fully labels an entire
mouse brain in just 24 hrs using proprietary stochastic electrotransport technology. SL additionally employs an
affinity ramp, a method in which antibodies are evenly distributed throughout the tissue before binding to target
proteins to produce labeling that is strikingly uniform in intensity from the sample’s surface to its core. During
Phase I, we broadened SL’s applications by (1) extending SL’s rapid immunolabeling capability for tissues
processed using iDISCO, (2) ensuring compatibility with key morphological, cell type, and neuronal activity
markers such as c-Fos, (3) adapting the technology to work with diverse sample types such as human cerebral
organoids, and (4) developing a prototype next-generation SL that performed simultaneous and cost-effective
cohort-level immunolabeling of multiple organoids or adult mouse brains. Having completed all the Phase I
project goals, we are now poised – in Phase II – to complete the development of the next generation SL, a dual
function clearing an...

## Key facts

- **NIH application ID:** 10414097
- **Project number:** 5R44MH121158-04
- **Recipient organization:** LIFECANVAS TECHNOLOGIES, INC.
- **Principal Investigator:** Katherine Cora Ames
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $350,347
- **Award type:** 5
- **Project period:** 2019-06-15 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10414097

## Citation

> US National Institutes of Health, RePORTER application 10414097, 3D molecular phenotyping of intact brain tissue via high-throughput active immunohistochemistry (5R44MH121158-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10414097. Licensed CC0.

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