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- Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging

# Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging

Du Z, Lin JR, Rashid R, Maliga Z, Wang S, Aster JC, et al.

Nature Protocols . 2019;14(10):2900-2930. DOI [10.1038/s41596-019-0206-y](https://doi.org/10.1038/s41596-019-0206-y). PMID 31534232. PMCID PMC6959005.

How to cite

### AMA

Du Z, Lin JR, Rashid R, Maliga Z, Wang S, Aster JC, et al. Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging. Nat Protoc . 2019;14(10):2900-2930. doi:10.1038/s41596-019-0206-y

### APA

Du, Z., Lin, J. R., Rashid, R., Maliga, Z., Wang, S., Aster, J. C., et al. (2019). Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging. Nature Protocols , 14(10), 2900-2930. https://doi.org/10.1038/s41596-019-0206-y

### BibTeX

@article{du2019qualifying,
title = {Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging},
author = {Du, Ziming and Lin, Jia-Ren and Rashid, Rumana and Maliga, Zoltan and Wang, Shu and Aster, Jon C. and others},
journal = {Nature Protocols},
volume = {14},
number = {10},
pages = {2900--2930},
year = {2019},
doi = {10.1038/s41596-019-0206-y},
pmid = {31534232}
}

Multiplexed tissue imaging can map dozens of proteins in a single tissue section, but the results are only as trustworthy as the antibodies behind them, and one poorly behaved antibody can quietly corrupt a whole panel.

This protocol sets out step by step how to confirm that an antibody is selective and specific before it enters a panel, and how to build and validate the panel itself. The authors demonstrate the workflow by assembling a 16-antibody immune-profiling panel that maps T cells, B cells, macrophages, and immune-checkpoint markers, imaging it cycle by cycle on a RareCyte CyteFinder microscope using tissue-based cyclic immunofluorescence (t-CyCIF).

[Read publication at Nature Protocols](https://pmc.ncbi.nlm.nih.gov/articles/PMC6959005/)

## Key findings

- The protocol is demonstrated by assembling a 16-antibody immune-profiling panel. Tissue-based cyclic immunofluorescence on FFPE tonsil and lung-cancer controls builds and validates a panel that enumerates and localizes T cells, B cells, macrophages, and cells expressing immune-checkpoint regulators.

- Imaging runs on a RareCyte CyteFinder capable of six-channel, whole-slide acquisition. The demonstrated panel used 4 of the 6 available channels per staining cycle, iterating antibody staining, imaging, and fluorophore inactivation to build marker depth across successive rounds.

- Every antibody is qualified on control tissues and cell lines before it earns a place in the panel. Fluorophore-conjugated antibodies start at a concentration of 10 µg/ml and are scored for subcellular localization, signal-to-noise ratio, and dynamic range, so unreliable reagents are caught before panel construction.

## CyteFinder in the methods

“In this study, we used the RareCyte CyteFinder, which is capable of six-channel, whole-slide imaging and precise slide positioning (note that only four channels were used for the work described here). We use Imager 5 software (RareCyte) for data acquisition.”

— Du et al., Nature Protocols (2019), Experimental design, “Imaging on a slide-scanning microscope”

Disclosure: RareCyte is named in the competing-interests statement of the publication cited above.

## Why it matters for CyteFinder users

If you run multiplexed tissue imaging, this is the protocol the field points to for getting antibodies right, and the RareCyte CyteFinder is the instrument it is written around. The authors designate the CyteFinder as the microscope their t-CyCIF workflow is optimized for, then use it to build and validate a 16-antibody immune panel one staining cycle at a time. Two CyteFinder capabilities carry that workflow: six-channel whole-slide imaging, so an entire section is captured rather than a hand-picked field, and precise slide repositioning, so the same cells line up across every cycle of staining, imaging, and fluorophore inactivation. Because cyclic immunofluorescence builds marker depth over successive rounds, registration and stable illumination are not niceties — they decide whether later cycles can be trusted against the first. For your own panels, that is the CyteFinder's role: a slide-scanning fluorescence microscope, paired with Imager 5 acquisition software, that holds position and signal steady long enough for antibody qualification and high-plex imaging to mean something.

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