Abstract
In situ hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate covisualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid and compatible with a broad range of optical systems. Here, we introduce a unified technical platform, termed ‘pSABER’, for the amplification of ISH signals in cell and tissue systems. pSABER decorates the in situ target with concatemeric binding sites for a horseradish peroxidase-conjugated oligonucleotide, enabling the localized deposition of fluorescent or colorimetric substrates. We demonstrate that pSABER effectively labels DNA and RNA targets in cultured cells and FFPE specimens. Furthermore, pSABER can achieve fivefold signal amplification over conventional signal amplification by exchange reaction (SABER) and can be serially multiplexed using solution exchange. Therefore, by linking nucleic acid detection to robust signal amplification capable of diverse readouts, pSABER will have broad utility in research and clinical settings.
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Data availability
Primary microscopy data, which have a substantial disk footprint, will be made available upon request. Source data are provided with this paper.
Code availability
The source code for the automated analysis of peak puncta intensities as well as example input images is available at https://github.com/beliveau-lab/SABER-Spot-Quant under an MIT license.
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Acknowledgements
We thank members of the Shechner, Shendure, Lieberman, Schweppe, Akilesh and Beliveau laboratories for helpful discussion about this work. We thank N. Peters of the UW W.M. Keck Microscopy Center and D. Fong of Nikon for assistance with microscopy during the development of pSABER and S. Jiang for helpful advice about working with HRP in tissues. This work was supported by a Damon Runyon Dale F. Frey Breakthrough Award (32-19 to B.J.B.), the National Institutes of Health (under grants 1R35GM137916 to B.J.B, 1UM1HG011586 to J.S. and B.J.B., 1R01DK130386 to S. Akilesh and 1R01GM138799-01 to D.M.S.), the Andy Hill Cancer Research Endowment (under a COVID-19 Response Grant Award to B.J.B. and S. Akilesh) and the Diabetic Complications Consortium (under grant 19AU3987 to S. Akilesh and B.J.B.). This project was also supported in part by a Building Bridges Award to J.A.L. and S. Akilesh from the Department of Laboratory Medicine and Pathology. A.F.T. was supported by NIH training grant T32GM007750.
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S. Attar, S. Akilesh and B.J.B. conceived the study. V.E.B. wrote and optimized software code. S. Attar, M.K., Y.L., V.E.B., E.K.N. and A.F.T. performed experiments. S. Attar, V.E.B., M.K., S. Akilesh and B.J.B. wrote the paper. All authors edited and approved the paper. D.M.S., J.S., D.K.S., J.A.L., S. Akilesh and B.J.B. supervised the work.
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S. Attar, A.F.T., D.M.S., S. Akilesh and B.J.B. have filed a patent application covering pSABER. B.J.B. is listed as an inventor on patent applications related to the SABER technology. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Visualization of PER extension products relevant to Fig. 4.
a, Gel electrophoresis shows the lengths of E1 and E2 from Fig. 4a. E1 is ~450 nt and E2 is ~700 nt. b, Gel electrophoresis shows the lengths of probes used for Fig. 4c. Probe length without the spacer is 65 nt. c, Gel electrophoresis shows the length of the mouse minor satellite PER used for Fig. 4e. Extension length is ~800 nt.
Extended Data Fig. 2 Quantification of the variability of HRP-oligo activity.
Absorbance values representing the activity of three HRP oligos over the course of three days.
Extended Data Fig. 3 Comparison of SABER and pSABER on a slide-scanning microscope.
a, SABER (left) and pSABER (right) visualization of mouse major satellite DNA imaged using a 10x air objective. b, SABER (left) and pSABER (right) visualization of mouse major satellite DNA imaged using a 20x air objective. c, SABER (left) and pSABER (right) visualization of mouse major satellite DNA imaged using a 10x air objective. Each image is presented at the indicated high (top) and low (bottom) contrast settings to allow for visual comparisons between signals occupying different portions of the 16-bit dynamic range (0–65535). Images are maximum projections in Z. Scale bars, 20 µm (insets) or 100 µm (fields of view). Each experiment was repeated ≥3 times and yielded similar results.
Extended Data Fig. 4 Efficiency and fidelity of Exchange-pSABER.
a, Schematic of Exchange-pSABER in which exchange occurred after hybridization with an HRP-oligo but before localized label deposition to assess exchange efficiency. b, Representative images of DNA pSABER targeting mouse minor satellite in EY.T4 mouse embryonic fibroblasts using three different combinations of HRP-oligo and fluorescent tyramide. The mock condition proceeded through pSABER as normal whereas the exchange condition developed after displacing the HRP-oligos. Images are displayed at the indicated high (top) and low (bottom) contrast settings to illustrate exchange efficiency. Scale bars, 10 µm. Each experiment was repeated ≥3 times and yielded similar results.
Extended Data Fig. 5 Biotin-ss-tyramide unlocks multiplexing for small targets.
a, Schematic of a pSABER experiment using biotin-ss-tyramide as the substrate and fluorescent streptavidin to visualize label deposition. Deposited biotin-ss-tyramide was reduced to remove the signal. b, Representative images of DNA pSABER targeting telomeres in HCT-116 CTCF-AID human colorectal cancer cells. The mock samples (left) incubated in PBS while the reduced (right) samples incubated in DTT. Images are displayed at the indicated high (top) and low (bottom) contrast settings to show the decrease in signal as a result of reduction. c, Images of DNA pSABER targeting alpha satellites in HCT-116 CTCF-AID human colorectal cancer cells comparing fluorescent streptavidin signal before and after reduction with DTT. d, Schematic of a pSABER experiment using biotin-ss-tyramide in which reduction occurred before fluorescent streptavidin staining to assess reduction efficiency. e, Images of DNA pSABER targeting telomeres in HCT-116 CTCF-AID human colorectal cancer cells showing reduction before staining (left) and reduction after staining (right), highlighting the effect bound fluorescent streptavidin imposes on reduction efficiency. Scale bars, 10 µm. Each experiment was repeated ≥3 times and yielded similar results.
Extended Data Fig. 6 Combinatorial labeling with fluorescent pSABER.
a, Schematic of a pSABER experiment depicting two rounds of labeling with different fluorescent tyramides. One target has a combination of hairpins, recruiting multiple HRP oligos throughout the experiment instead of one. b, Images of DNA pSABER targeting four 200-kb spots on chromosome X in a male human metaphase spread. The white box in the left image indicates the zoomed in field of view. The red arrowhead marks spot 17, which was labeled with two colors. Scale bar, 20 µm. Each experiment was repeated ≥3 times and yielded similar results.
Supplementary information
Supplementary Data 1
Sequences of oligonucleotides used in the study.
Source data
Source Data Fig. 4
Source numerical data plotted in Fig. 4b,d,f.
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Attar, S., Browning, V.E., Krebs, M. et al. Efficient and highly amplified imaging of nucleic acid targets in cellular and histopathological samples with pSABER. Nat Methods 22, 156–165 (2025). https://doi.org/10.1038/s41592-024-02512-2
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DOI: https://doi.org/10.1038/s41592-024-02512-2
