Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues

Abstract

Spatial mapping of proteins in tissues is hindered by limitations in multiplexing, sensitivity, and throughput. Here we report immunostaining with signal amplification by exchange reaction (Immuno-SABER), which achieves highly multiplexed signal amplification via DNA-barcoded antibodies and orthogonal DNA concatemers generated by primer exchange reactions (PER). SABER offers independently programmable signal amplification without in situ enzymatic reactions, and intrinsic scalability to rapidly amplify and visualize a large number of targets when combined with fast exchange cycles of fluorescent imager strands. We demonstrated 5–180-fold signal amplification in diverse samples (cultured cells, and FFPE, cryosectioned or whole mount tissues), and simultaneous signal amplification for 10 different proteins using standard equipment and workflows. We also combined SABER with expansion microscopy to enable rapid, multiplexed super-resolution tissue imaging. Immuno-SABER presents an effective and accessible platform for multiplexed and amplified imaging of proteins with high sensitivity and throughput.

Light microscopy based approach for mapping connectivity with molecular specificity

“…Probing multiple proteins using traditional immunohistochemistry (IHC) is typically limited by host animal species of primary antibodies (most tend to be either mouse or rabbit) and visible light bandwidth, such that detecting more than 4 targets becomes difficult. Recent strategies, such as Immuno-SABER 16 , PRISM 17 , and CODEX 18 have been developed to overcome these limitations using antibody-DNA barcoding and read out strategies. Alternatively, tissue sections can undergo multiple rounds of routine antibody staining, imaging, stripping, and restaining, as seen in array tomography 19 , CLARITY 20 , MAP 21 , and SHIELD 22 .…”

“…That said, we expect a more photon efficient volumetric imaging modality, such as light sheet microscopy, will yield better signal to noise under the same labeling conditions than confocal microscopy used in our study. Moving forward, it will be important to optimize FP expression along axonal membranes, as well as explore alternative signal amplification technologies, such as immuno-SABER 16 .…”

CytoMAP: a spatial analysis toolbox reveals features of myeloid cell organization in lymphoid tissues

“…histo-cytometry, CODEX, MIBI, STARMAP). 4,[10][11][12][13][14][15][16][17] These techniques generate panoptic datasets describing phenotypic, transcriptional, functional, and morphologic cellular properties, while retaining information on the precise 2-dimensional (2D) or 3D positioning of cells within tissues, thus potentiating a more complete understanding of cellular function directly in situ. Analysis of images generated by these methods requires multiple processing steps, including image preprocessing, cell segmentation, object classification, and spatial analysis.…”

Online narrative guides for illuminating tissue atlas data and digital pathology images

“…These include Imaging Mass Cytometry (IMC) 1 and Multiplexed Ion Beam Imaging (MIBI) 2 , which detect antigens using metal isotope-labeled antibodies, tissue ablation, and atomic mass spectrometry. In contrast, methods such as MxIF 3 , CODEX 7 , t-CyCIF 4 , multiplexed IHC 8 , and immuno-SABER 9 , use fluorescently-labelled (or enzyme-linked) antibodies followed by imaging on a fluorescence microscope. These methods differ in the number of antigens they can routinely detect on a single tissue section (currently ~12 in the case of multiplexed IHC to ~40-60 in the case of IMC or t-CyCIF); some methods are restricted to selected fields of view (typically a ~500 µm square) whereas others can perform whole slide imaging (WSI) on an area ~400-1,000 times larger.…”