A new method to simultaneously visualize the location of numerous proteins within human cells and tissues with high resolution has been developed. Called Immunostaining with Signal Amplification By Exchange Reaction, or Immuno-SABER, it has the potential to accelerate many large-scale protein mapping and biomarker discovery projects, according to a team from Harvard’s Wyss Institute.
The approach, described today in Nature Biotechnology, combines the protein targeting specificity of commonly available antibodies with a DNA-based signal-amplification strategy that enables the highly multiplexed visualization of many proteins in the same sample with pre-programmable and tunable fluorescence signals at each target site.
"We demonstrated that Immuno-SABER provides the capability to independently tune the signal intensity for individual protein targets 5 to 180-fold, with multiplexing capability to allow the simultaneous detection of many proteins. Together with its speed, relative ease of use, and low costs, this technique has the potential to fast-forward ongoing large-scale protein-mapping studies and biomarker discovery efforts across many tissues and diseases," said Peng Yin who is a Wyss Institute Core Faculty member.
Conventional antibody staining methods typically allow only a maximum of five different stains to be used simultaneously, and target proteins can differ significantly in their abundances, making it difficult to distinguish rare protein targets with high sensitivity from the background fluorescence that many tissues display. Immuno-SABER utilizes the "Primer Exchange Reaction" (PER) method previously reported by Yin's group to synthesize long concatemers of short DNA primer sequences with the help of a catalytic DNA hairpin structure. The PER-generated concatemers are attached via short handle sequences to DNA-barcodes on antibodies that bind to target proteins in fixed cell and tissue samples with high specificity. At the target site, SABER concatemers provide a scaffold with multiple binding sites for complementary fluorescent oligonucleotides ("imagers"), and thus a means to amplify the signal emanating from each protein target.
"By barcoding antibodies with unique short DNA sequences and applying Immuno-SABER, we can simultaneously visualize multiple protein targets on the same sample and with high specificity. This essentially opens up a way to analyze the protein variety present in tissues in a robust and multiplexed fashion," said co-first and co-corresponding author Sinem Saka, Ph.D.
Image: A cryosection of the mouse retina in which the researchers visualized 10 proteins at a time with Immuno-SABER's multiplexing abilities. Image courtesy of Wyss Institute at Harvard University.