2. INTRODUCTION
Immunostaining is an immunofluorescence
technique allowing the visualization of the
specific protein or antigen in a tissue sections
by binding a specific antibody chemically
conjugated with the fluorescent dye.
The immunofluorescence technique was
firstly described in 1942 and refined by Coons
in 1950, which used a fluorescence
microscope able to read the specific
immunological reaction on tissue or cell
prepared on slides.
3. Immuno-SABER
• SABER- Signal Amplification By Exchange Reaction.
• New method to simultaneously visualize the location of numerous proteins within animal cells and
tissues with high resolution.
• It has potential to accelerate many large scale protein mapping and biomarker discovery projects.
• The approach described in Nature Biotechnology.
• It combines the protein targeting specificity of commonly available antibodies with DNA based
signal amplification strategy.
• It enables the highly multiplexed visualization of many protein in the same sample with pre
programable and amplified signals at each target site.
4. This is the first step of reaction Primer
Exchange Reaction to synthesize a longer
concatemer of identical short sequences.
5. This is the DNA tagged antibody the primer
end work as a handle that is complementary
to a DNA tag attached to an antibody.
6. The DNA tagged antibody first attached to
the target protein and in second step the
PER concatemer is bind via its handle to
hybridised DNA tagged antibody at the
protein target site.
7. The PER concatemers provide scaffolds onto
which multiple fluorescent imagers with
short DNA sequence that are complementary
to the concatemer repeats can be assembled.
9. The branched concatemers that are binding to
internal sites of already existing PER
concatemers attached to DNA tagged antibody
can incorporate a significantly higher number of
fluorescent and provide greater sensitivities.
10. A cell is visualised with a linear PER
concatemer and a branched concatemer on
right.
11. The combination of different proteins inside the
cell using fluorescent imager that emit light at
different wavelengths corresponding to different
colours.
12. Simultaneous visualisation of different
protein target with different wavelength of
light in a cell or tissue section.
13. SABER Exchange in this one set of
fluorescent imagers bound to PER
concatemers at a series of protein target sites
is captured and then washed out of the sample
and replaced by another set of fluorescent
imagers that bind to PER concatemer at
different sites.
14. Exchange SABER at different protein site. This
exchange reaction can be replaced multiple
times.
15. In this example four different proteins are
simultaneously detected in a cell with
immune SABER with the help of four
different antibodies attached per concatemers.
16. Final fluorescent of four different protein in a
cell with different fluorescence colour.
20. Signals were quantified in each
case versus the unamplified
sample and the fold changes
are provided.
21. Signal intensity for each nucleus was
obtained from automated
segmentation and the histogram was
plotted for the whole tissue section
for each condition.
