TITLE: SABER-FISH – Signal amplification for multiplexed fluorescence in situ hybridization assays
AUTHORS: 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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">The SABER Technology</span></div><div class = "text-block"><span>The Signal Amplification By Exchange Reaction (SABER) method is used for amplifying signal from multiplexed </span><span style = "font-style:italic;">in situ </span><span>fluorescence staining experiments. Developed by the Yin and Cepko labs at Harvard University and the Wyss Institute, the technique uses </span><a href="https://www.nature.com/articles/nchem.2872" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Primer Exchange Reactions (PERs)</span></a><span> to generate three-letter (A, T, C) concatemeric sequences in bulk </span><span style = "font-style:italic;">in vitro </span><span>reactions. These concatemers can then be </span><span style = "font-style:italic;">in situ </span><span>hybridized to fixed cells and tissues and act as scaffolds that localize fluorescent 'imager' strands. The method can further be paired with </span><a href="https://pubs.acs.org/doi/10.1021/acs.nanolett.7b02716" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">DNA-Exchange Imaging (DEI)</span></a><span> 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. </span></div><div class = "text-block"><span>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 (</span><a href="https://www.nature.com/articles/s41592-019-0404-0" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">SABER-FISH</span></a><span>) and protein targets (</span><a href="https://www.nature.com/articles/s41587-019-0207-y" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Immuno-SABER</span></a><span>, </span><a href="http://immuno-saber.net" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">website</span></a><span>, </span><a href="https://www.protocols.io/private/8AB389FCC03811EAA8870A58A9FEAC2A" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">collection on protocols.io</span></a><span>).</span></div><div class = "text-block"><span style = "font-weight:bold;">SABER-FISH</span></div><div class = "text-block"><span>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 </span><span style = "font-style:italic;">in situ </span><span>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 </span><span style = "font-style:italic;">in vitro </span><a href="https://www.nature.com/articles/nchem.2872" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Primer Exchange Reactions (PERs)</span></a><span> 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 </span><span style = "font-style:italic;">in situ </span><span>hybridized to fixed cell and tissue samples, followed by a short secondary fluorescent hybridization that binds 20nt fluorophore-conjugated strands ('imagers') to the concatemers.</span></div><div class = "text-block"><span>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 </span><a href="https://pubs.acs.org/doi/10.1021/acs.nanolett.7b02716" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">DNA-Exchange Imaging (DEI)</span></a><span>. SABER-FISH signal can further be enhanced with a branching strategy, where multiple rounds of concatemerized probe binding create branched structures </span><span style = "font-style:italic;">in situ</span><span>. Below are a number of resources intended to help adoption of the technology, including references, animations, and protocols.</span></div></div>

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