Collection Citation: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin 2020. SABER-FISH – Signal amplification for multiplexed fluorescence in situ hybridization assays. protocols.io https://dx.doi.org/
Manuscript citation:
Kishi, J.Y., Lapan, S.W., Beliveau, B.J. et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat Methods 16, 533–544 (2019). https://doi.org/10.1038/s41592-019-0404-0
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: July 06, 2020
Last Modified: August 21, 2020
Collection Integer ID: 38918
Keywords: multiplexed fluorescence, signal amplification for multiplexed fluorescence, multiplexing with saber, fluorescence imaging, iteritave rounds of fluorescence imaging, fish multiplexed signal amplification, signal amplification in fluorescence, short secondary fluorescent hybridization, saber technology the signal amplification, situ fluorescence, fluorophores on complementary imager, situ stainings for rna, hybridization of new imager strand, fluorescence, saber technology, expensive microscopy setup, new imager strand, separated fluorophore, saber, tissue sample, concatemerized probe, staining experiment, complementary to dna, cell basis for quantitative analysis, imager exchange, rapid tissue mapping, old imager strand, fluorescent, exchange imaging, multiplexed signal amplification, situ hybridization, vitro reaction, probe binding, fish signal, cell segmentation, transcriptomic loci, underlying probe binding, cell segmentation with puncta counting
Abstract
The SABER Technology
The Signal Amplification By Exchange Reaction (SABER) method is used for amplifying signal from multiplexed in situ fluorescence staining experiments. Developed by the Yin and Cepko labs at Harvard University and the Wyss Institute, the technique uses Primer Exchange Reactions (PERs) to generate three-letter (A, T, C) concatemeric sequences in bulk in vitro reactions. These concatemers can then be in situ hybridized to fixed cells and tissues and act as scaffolds that localize fluorescent 'imager' strands. The method can further be paired with DNA-Exchange Imaging (DEI) to increase multiplexing via rapid stripping of old imager strands and hybridization of new imager strands (imager exchange) and/or cell segmentation with puncta counting on a per cell basis for quantitative analyses.
See the following references and resources below for further information. SABER provides a scalable and cost-effective way to amplify multiplexed in situ stainings for RNA/DNA (SABER-FISH) and protein targets (Immuno-SABER, website, collection on protocols.io).
SABER-FISH
Multiplexed signal amplification enables rapid tissue mapping by increasing the number of targets that can be visualized per sample and reducing the exposure times required to see signals. Signal amplification in fluorescence in situ hybridization (FISH) assays can also increase sensitivity, potentially allowing smaller genomic and transcriptomic loci to be imaged, fewer probes to be applied, and/or less expensive microscopy setups to be utilized. SABER-FISH uses in vitro Primer Exchange Reactions (PERs) to synthesize long repetitive 'concatemer' sequences onto the 3' end of probes designed to be complementary to DNA and RNA targets of interest. After concatemer extension, probes are in situ hybridized to fixed cell and tissue samples, followed by a short secondary fluorescent hybridization that binds 20nt fluorophore-conjugated strands ('imagers') to the concatemers.
Multiplexing with SABER-FISH is achieved through the use of orthogonal concatemer sequences appended to probe sets, which can be read out to spectrally separated fluorophores on complementary imagers. Imager sequences can also be stripped from one set of concatemers without disrupting the underlying probe binding, which allows new sets of imagers targeting different loci to be imaged in iteritave rounds of fluorescence imaging. This process of stripping and hybridization is referred to as DNA-Exchange Imaging (DEI). SABER-FISH signal can further be enhanced with a branching strategy, where multiple rounds of concatemerized probe binding create branched structures in situ. Below are a number of resources intended to help adoption of the technology, including references, animations, and protocols.
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