Method Article
A Hybridization Chain Reaction-based Method for Amplifying Immunosignals
https://doi.org/10.1038/protex.2018.014
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We describe the development of immunosignal HCR (isHCR), which combines antibody-antigen interactions with Hybridization Chain Reaction technology for amplifying immunofluorescence by up to 2-3 orders of magnitude, and with low background. We further optimized its use in multiplexed imaging and in state-of-the-art tissue expansion and clearing techniques. With its highly modular and easily adaptable design, isHCR can be applied broadly in basic research and clinical diagnostics.
Immunofluorecence
Neuroscience
Cell Biology
Hybridization Chain Reaction
Multiplexed Labeling
Antibody-based immunoassays remain the most popular methods for detecting and identifying the location of proteins and other biomolecules in biological samples. A major limitation in the use of immunoassays is that the low abundance of a given target molecule in a sample often necessitates signal amplification before detection is possible. Amplification can be achieved using conjugated enzymes. Current amplification methods have several drawbacks: 1) high background, 2) reduced spatial resolution, 3) lack of multiplexity, and 4) unsuitable for use with large-volume samples in new tissue expansion and clearing techniques. To overcome many of these limitations, we adapt hybridization chain reaction (HCR) technology to amplify immunosignals. We here describe immunosignal HCR (‘isHCR’) and demonstrate the use of HCR in a variety of immunoassays. The streptavidin-biotin interaction can be utilized for the typical isHCR application. isHCR uses label-free streptavidin, which allows the attachment of synthesized 5’- biotinylated DNA HCR initiators to the biotinylated antibodies. These initiators can then be elaborated with fluorescent DNA HCR amplifiers for signal detection. Alternatively, the DNA HCR initiators can be conjugated directly onto antibodies, therefore omitting the streptavidin-biotin interaction.
1x Phosphate Buffered Saline (PBS)
TritonX-100
5× sodium chloride citrate (SCC buffer)
2% Paraformaldehyde (PFA) in PBS
Tween-20
dextran sulfate
BSA
Graphene Oxide
Streptavidin
DNA HCR initiator
DNA HCR amplifiers (the sequences and modifications can be found in the associated publication)
thermal cycler, fluorescent microscopy
Tissue section preparation
Immunostaining
Wash and permeablize sections in 0.3% Triton X-100 in PBS (PBST) and blocked them in 2% BSA in PBST at room temperature for 1 h.
Incubate sections with primary antibodies for various times according to the efficiency of the antibodies.
Wash samples for three times in PBST. (if DNA HCR initiator conjugated secondary antibodies are used, incubate samples in HCR amplification buffer [5× sodium chloride citrate (SCC buffer), 0.1% vol/vol Tween-20, and 10% wt/vol dextran sulfate in ddH2O] for 30min at room temperature)
Sections are incubated with biotinylated secondary antibodies or DNA HCR initiator conjugated secondary antibodies.
Wash samples for three times in PBST.
isHCR amplification
(All reagents were dissolved in HCR amplification buffer [5× sodium chloride citrate (SCC buffer), 0.1% vol/vol Tween-20, and 10% wt/vol dextran sulfate in ddH2O].)
(For biotinylated secondary antibodies, perform the following four steps first:
Incubate samples in 1 µg/mL streptavidin at room temperature for 30 min.
Wash three times in PBST.
Incubate samples with 0.5 µM DNA-biotin HCR initiators at room temperature for 30 min.
Wash three times in PBST.)
Snap-cooled a pair of DNA-fluorophore HCR amplifiers separately in 5× SSC buffer by heating at 95°C for 90s and cooling to room temperature over 30 min.
Both of these amplifiers were then added to amplification buffer to a final concentration of 12.5 nM (Optional: graphene oxide (GO) can be added for applications that demands background suppression. Add GO to the buffer from step 2 to 20 µg/mL and vortex thoroughly)
Incubate samples with the buffer from step 2 overnight at room temperature.
Wash three times in PBST.
mount sections on slides and observe.
M.L. is supported by China MOST (2012YQ03026005, 2013ZX0950910, 2015BAI08B02), NNSFC (91432114, 91632302), and the Beijing Municipal Government.
The authors declare no competing financial interests.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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