# Live-cell intracellular immunofluorescence

> **NIH NIH R21** · UNIVERSITY OF WASHINGTON · 2021 · $181,741

## Abstract

ABSTRACT
Immunofluorescence (IF) is one of the most important tools for both basic biology research and clinical sample
imaging due to the broad availability of antibodies and the resolution and sensitivity of fluorescence imaging.
Despite the tremendous amount of data produced by IF in the past ~60 years since its initial invention, IF suffers
from a fundamental limitation: incapability of interrogating intracellular targets in live cells (cells have to be
fixed first, thus only providing a snapshot of the dynamic cell signaling process). Considering the 3D structure of
cells, there are far more intracellular targets with biological significance than their cell-membrane counterparts,
but immunological agents such as antibodies, antibody fragments, peptides, nanobodies, and scFvs, being
highly hydrophilic macromolecules, cannot spontaneously cross the cell membranes. To overcome the barriers
of cell membrane and endocytosis that are extremely effective in preventing intact biomolecules to enter the
cytosol, we propose to develop an intracellular protein delivery technology building on a unique concept: non-
covalent cholesterol tagging. In contrast to conventional delivery technologies that are mostly based on
endocytosis (inappropriate for live cell imaging due to the high background generated by imaging agents trapped
in endosomes), our small-molecule tag enables proteins to permeate through the cell membrane. More
importantly, this is achieved without generating pores in the membrane that cause cytotoxicity. If successful, this
platform technology should open a whole new dimension for molecular biology, drug development, and cell
engineering.

## Key facts

- **NIH application ID:** 10285673
- **Project number:** 1R21CA263698-01
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Xiaohu Gao
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $181,741
- **Award type:** 1
- **Project period:** 2021-08-10 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10285673

## Citation

> US National Institutes of Health, RePORTER application 10285673, Live-cell intracellular immunofluorescence (1R21CA263698-01). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10285673. Licensed CC0.

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