Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging

Abstract

Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.

Fig. 1 |. obtaining high-content imaging data using a wide range of multiplexed antibody-based imaging platforms.

a, Fifty-plex confocal images of a human mesenteric lymph node obtained by the IBEX method. Two to four marker overlays for two regions (germinal center, white rectangle and medullary cords, red rectangle) are shown in higher zoom. Scale bars, 500 and 100 μm, for the overview and zoom-in images, respectively. β-Tubulin 3 (β-Tub3), collagen IV (Coll IV), fibronectin (Fibro), laminin (Lamin), and vimentin (Viment) (original lymph node dataset from Radtke et al.). b, Graphical representation of the main approaches for multiplexed antibody-based imaging. Antibodies are commonly labeled with metals, fluorophores, or DNA oligonucleotides for complementary binding of fluorescently tagged DNA probes.

Fig. 2 |. Considerations for the choice and implementation of multiplexed antibody-based imaging technologies into existing workflows.

a, Open-source, commercial, and core facility options can be separated by advantages and disadvantages related to ease of implementation, initial cost investment, cost per experiment, flexibility and customization, and the required expertise. b, Several factors govern which method to implement: imaging data requirements (area of tissue needed to be imaged per sample, resolution of final images, time required for imaging each sample, number of markers), sample requirements (number and format of samples, preservation method used for samples, tissue autofluorescence, and whether 2D or 3D volume data are needed), and infrastructure requirements (where existing equipment can be leveraged, level of bioinformatics needed for analysis, technical support, and whether reagents can be purchased or must be customized). Comparisons between different multiplexed imaging techniques are summarized in Supplementary Table 1 and have been described in greater detail in other reviews^,,.

Fig. 3 |. Phases of panel development and validation for multiplexed antibody-based imaging assays.

Graphical representation of assay development. In Step 1, markers are selected on the basis of the indicated criteria. Some multiplexing methods require custom reagents (that is, directly conjugated primary antibodies); see Fig. 4 for more details. In Step 2, antibodies are validated individually to verify target specificity. In Step 3, the full panel is validated to ensure that inclusion of additional antibodies does not affect target specificity. In Step 4, data are collected and analyzed.

Fig. 4 |. Process of conjugating antibodies with modifiers for multiplexing.

Following the selection of labeling chemistry and crosslinker, the workflow typically consists of these steps: (1) activation of modifier groups (for example reducing of thiol groups) and buffer exchange, (2) reaction with the crosslinker, (3) removal of the excess crosslinker, and (4) reaction of the antibody with the modifier. Since the reaction typically includes an excess of the modifier, the unreacted molecules are optionally removed from the mixture using buffer exchange, gel filtration, or sequence-directed pull-downs in the case of oligonucleotide barcode modifiers. Further purification can be performed by ion exchange or size-exclusion chromatography to remove the aggregated or degraded molecules, leftover modifiers, and under- or overconjugated antibodies. While purified reagents often improve quality and performance, purification may substantially reduce conjugated antibody yield, making it impractical or costly for small-scale preparations. The final product and its purity can be validated by a band shift on SDS–PAGE gels, where both the antibody and the modifier can be observed (for example using Coomassie, fluorescence, Sybr stains, or silver stain). The resulting antibody concentration can be determined using bicinchoninic acid assay (BCA) or spectrophotometry (for example, NanoDrop), although crosslinkers may interfere with these absorbance measurements and corrections need to be made accordingly. Crucially, the final product should be revalidated for the general target binding specificity (for example by flow cytometry) and for the assay of interest by functional comparison to the unconjugated antibody using direct detection or secondary antibody detection.