MULTIPLEXED IMMUNOSIGNAL AMPLIFICATION USING HYBRIDIZATION CHAIN REACTION-BASED METHOD

Abstract

The invention provides a method for optimizing isHCR for multiplexed labeling, which combines binder-biomolecule interactions with hybridization Chain Reaction (HCR).

Claims

1. A method for detecting multiple target biomolecules, which combines binder-biomolecule interactions with Hybridization Chain Reaction (HCR), comprising: targeting the multiple target biomolecules with orthogonal HCR initiators; and directing the orthogonal HCR initiators to orthogonal binders for conjugating orthogonal HCR initiators, wherein the directed orthogonal HCR initiators are used in HCR to allow HCR amplification of multiple target biomolecules with orthogonal pairs of HCR amplifiers, wherein each of the orthogonal HCR initiators has a region for hybridizing with a HCR amplifier, and a region for conjugating a binder, and the orthogonal binders target multiple target biomolecules respectively to allow HCR amplification directed to multiple target biomolecules, wherein the orthogonal binders are orthogonal antibodies, orthogonal nanobodies, or orthogonal antibody fragments of antibodies, wherein the orthogonal HCR initiators are directly conjugated to the orthogonal binders using chemical linkers, the chemical linker is a click chemistry linker which is selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker, and wherein the orthogonal HCR initiators are conjugated to the orthogonal antibodies by reacting DBCO-labeled HCR initiators with Azide-activated binders.

2. The method of claim 1, wherein the orthogonal antibodies, orthogonal nanobodies, or orthogonal antibody fragments of antibodies are a IgG, nanobody or scFv.

3. The method of claim 1, wherein the pair of amplifiers are terminally modified or internally modified with a chemical group or a fluorescent dye, which allows initiating further rounds of amplification, and wherein the chemical group is selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazine, Alkyne, or DBCO, and the fluorescent dye is selected from FITC, Cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluro 546, Alexa Fluor 488, and Alexa Fluor 647.

4. The method of claim 3, wherein the pair of amplifiers are modified at internal positions, which are accessible to streptavidin and which serve as anchors for each successive round of branching in multi-round isHCR.

5. The method of claim 1, further comprising using grapheme oxide (GO) for absorbing unassembled HCR amplifiers; or further comprising using grapheme oxide (GO) for absorbing unassembled HCR amplifiers and quenching the fluorescence, wherein the amplifiers are terminally modified or internally modified with fluorescent dye.

6. The method of claim 5, wherein GO has a particle size of <500 nm.

7. A kit for detecting multiple target biomolecules, which comprises: (1) orthogonal binders; (2) orthogonal HCR initiators; and (3) orthogonal pairs of HCR amplifiers, wherein each of HCR initiators has a region for hybridizing with a HCR amplifier, and a region for conjugating a binder, and the orthogonal binders target multiple target biomolecules respectively to allow HCR amplification directed to multiple target biomolecules, wherein the orthogonal binders are orthogonal antibodies, orthogonal nanobodies, or orthogonal antibody fragments of antibodies, wherein the orthogonal HCR initiators are directly conjugated to the orthogonal binders using click chemistry linkers which are selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker, and wherein the orthogonal HCR initiators are conjugated to the orthogonal antibodies by reacting DBCO-labeled HCR initiators with Azide-activated binders.

8. The kit of claim 7, wherein the antibody is a IgG, nanobody or scFv.

9. The kit of claim 7, wherein an amplifier or a pair of amplifiers are terminally modified or internally modified with a chemical group or a fluorescent dye, which allows initiating further rounds of amplification, and wherein the chemical group is selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazine, Alkyne, or DBCO, and the fluorescent dye is selected from FITC, Cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluro 546, Alexa Fluor 488, and Alexa Fluor 647.

10. The kit of claim 9, wherein the pair of amplifiers comprise modification at internal positions, which are accessible to streptavidins and which serve as anchors for each successive round of branching in multi-round isHCR.

11. The kit of claim 7, further comprising grapheme oxide (GO) for absorbing unassembled HCR amplifiers; or further comprising grapheme oxide (GO) for absorbing unassembled HCR amplifiers and quenching the fluorescence, wherein the amplifiers are terminally modified or internally modified with fluorescent dye.

12. The kit of claim 11, wherein GO has a particle size of <500 nm.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1. Multiplexed labeling using isHCR.

[0022] FIG. 2. Simultaneous detection of multiple targets using isHCR.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0023] In the first embodiment, we established the use of two biotinylated secondary antibodies in combination with two orthogonal DNA HCR initiators, which allows isHCR to sequentially amplify two targets in the same brain section sample (FIG. 1a).

[0024] In the second embodiment, we directly conjugated DNA HCR initiators to secondary antibodies via SMCC or NHS-Azide linkers (FIG. 2a). This modification allows simultaneous isHCR amplification of multiple targets. We successfully performed multiplexed isHCR amplification using initiator-labeled secondary antibodies in western blotting (FIG. 2b), immunostaining of cultured cells (FIG. 2c), and immunostaining of brain sections (FIG. 2d).

[0025] In the third embodiment, with the goal of expanding the modularity of the isHCR platform yet further, we tested whether a variety of genetically-engineered protein tags could be added to target proteins in cells to enable the direct binding of targets to HCR initiators. Three orthogonal tags targeting different cellular locations (SpyTag for cell nuclei, SNAP-tag for mitochondria, and smFP_GCN4 for cell membranes) were expressed in cultured cells. DNA HCR initiators were conjugated to tag binding partners (SpyCatcher, benzylguanine (BG), and scFv), and these were subsequently used for isHCR amplification to detect the subcellular localization of the genetically-encoded tags (FIG. 2e). Strong and correctly localized signals were observed for each tag.

[0026] In the fourth embodiment, we next expressed membrane-bound GFP in brains and confirmed that HCR initiators conjugated to GFP nanobodies22 could bind directly to GFP, allowing for subsequent polymerization and detection of HCR amplifiers in brain sections (FIG. 1b).

[0027] In the fifth embodiment, we expressed the SNAP-tag in mouse brains and applied BG-functionalized HCR initiators for direct detection and amplification (FIG. 1c, d). We noted that labeling neurons with a monomeric SNAP-tag only generated weak signals at soma (FIG. 1d middle panel). We therefore employed a tandem SNAP-tag to enhance the labeling intensity. This optimization greatly increased the signal intensity and allowed for the detection of distal axons of labeled neurons (FIG. 1d bottom panel).

[0028] HCR is the abbreviation of Hybridization Chain Reaction. When a single-stranded DNA initiator is added to a reaction system, it opens a hairpin of one species (H1 amplifier), exposing a new single-stranded region that opens a hairpin of the other species (H2 amplifier). This process, in turn, exposes a single-stranded region identical to the original initiator. The resulting chain reaction leads to the formation of a nicked double helix that grows until the hairpin supply is exhausted.

[0029] isHCR in the present invention combines binder-biomolecule interaction with hybridization Chain Reaction (HCR), wherein the binder may be an antibody or a genetically-engineered protein tag for labeling a target biomolecule.

[0030] Click chemistry is a class of biocompatible reactions intended primarily to join substrates of choice with specific biomolecules. Click chemistry is not a single specific reaction, but describes a way of generating products that follow examples in nature, which also generates substances by joining small modular units. In general, click reactions usually join a biomolecule and a reporter molecule. Click chemistry is not limited to biological conditions: the concept of a “click” reaction has been used in pharmacological and various biomimetic applications. However, they have been made notably useful in the detection, localization and qualification of biomolecules.

[0031] Antibody in the present invention includes but not limited to traditional IgGs and nanobodies.

EXAMPLES

[0032] Methods and Materials

[0033] Reagents and reagent preparation. DNA oligos were synthesized by Thermo Fisher Scientific and Sangon Biotech. Detailed sequences and modifications of DNA oligos can be found in Table 1. All oligos were dissolved in ddH.sub.2O and stored at −20° C. Benzylguanine (BG)-labeled oligos were prepared by first mixing NH.sub.2-Oligo (2 mM, 4 μL), HEPES (200 mM, 8 μL; pH=8.5), and BG-Gal-NHS (20 mM in DMSO, 12 μL; 591515, NEB) for 30 min at room temperature, and then purified using Micro Bio-Spin P-6 Gel columns (7326221, Bio-Rad).

[0034] The detailed information for antibodies and fluorescent reagents is shown in Table 2. Dextran sulfate (D8906) were purchased from Sigma-Aldrich. Graphene Oxide (GO, XF020, particle size <500 nm, C/O ratio=1.6) was obtained from Nanjing XFNANO.

[0035] Plasmid construction and AAV packaging. The genes encoding SNAPf, SpyCatcher, and GFP nanobody (LaG-16-2) were synthesized according to original reports. 4×SNAPf sequence was assembled by fusing four SNAPf-encoding sequences with short peptide linkers using Gibson cloning. scFv-GCN4-HA-GB1 sequence was amplified from pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE (Addgene plasmid #60906, a gift from Ron Vale). The amino acid sequence smFP_GCN4 was designed based on the originally-reported smFPs sequence (Table 3). For membrane targeting, a GAP43-palmitoylation sequence was added by PCR to the 5′ end of GFP, SNAPf, and smFP_GCN4 (hereafter named mGFP, mSNAPf and msmFP_GCN4). Two tandem mitochondria targeting sequences from human Cox8a were amplified by PCR from genomic DNA of HeLa cells, and added to the 5′ end of SNAPf by Gibson assembly (hereafter named mitoSNAP). H2B and human GBP1 sequence was amplified by PCR from genomic DNA of HeLa cells. A single SpyTag sequence was added to the 3′ end of H2B by PCR (hereafter named H2B-SpyTag). mGFP, mitoSNAP, H2B-SpyTag, and msmFP_GCN4 were cloned into the pcDNA3.1 vector. scFv-GCN4-HA-GB1 and SpyCatcher were cloned into the pET-21a vector for bacterial cytosolic expression. LaG-16-2 was cloned into the pET-22b vector for bacterial periplasmic expression. AAV-DIO-mGFP was constructed as previously described. AAV-DIO-mSNAPf and AAV-DIO-4×SNAPf were constructed by inserting the sequences encoding mSNAPf or 4×SNAPf, in an inverted orientation, into an AAV-EF1a-DIO backbone derived from AAV-EF1α-DIO-hChR2(H134R)-mCherry (a gift from Karl Deisseroth). AAV vectors were packaged into the AAV2/9 serotype, with titers of 1-5×10.sup.12 viral particles □mL.sup.−1.

[0036] Purification of recombinant protein. E. coli BL21 (DE3) cells harboring pET-21a-scFv-GCN4-HA-GB1 or pET-21a-SpyCatcher were grown in lysogeny broth (LB) medium supplemented with 100 μg□mL.sup.−1 ampicillin. Protein expression was induced with IPTG at a concentration of 0.1 M for 3 h at 37° C.

[0037] Cells were then pelleted by a 20-min spin at 2,000×g at 4° C. Cells were lysed via ultrasonic sonication. Cellular debris was removed via 1 h of centrifugation at 39,000×g at 4° C. The supernatant was bound to His-Select nickel affinity resin, washed with His-wash buffer (20 mM NaH.sub.2PO.sub.4, pH 8.0, 1 M NaCl, 20 mM imidazole), eluted with His-elution buffer (20 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 250 mM imidazole), and the eluate was then dialyzed with phosphate buffer saline (PBS).

[0038] LaG-16-2 was expressed and purified according to the original report. In brief, E. coli BL21 (DE3) cells harboring pET-22b-LaG-16-2 were grown in LB medium supplemented with 100 μg□mL.sup.−1 ampicillin. Protein expression was induced with IPTG at a concentration of 0.1 M for 20 h at 12° C. Cells were pelleted via a 10-min of centrifugation at 5,000×g at 4° C. The periplasmic fraction was isolated by osmotic shock. This fraction was then bound to His-Select nickel affinity resin and purified as described above.

[0039] Protein-HCR DNA Initiator conjugation. The conjugation was performed using Maleimide-PEG2-NHS (SMCC, 746223, Sigma-Aldrich) or NHS-Azide (synthesized or purchased from Thermo Fisher Scientific, 26130) as linkers. For Maleimide-PEG2-NHS conjugation, proteins (IgGs, scFv, LaG-16-2 and SpyCatcher) were dialyzed into phosphate buffered saline (PBS, pH 7.4) and reacted with Maleimide-PEG2-NHS (7.5-fold molar excess) at room temperature for 2 h. Excess crosslinkers were removed from maleimide-activated proteins using Zeba spin columns (7000 MWCO). In parallel, thiol-modified HCR initiators were reduced using dithiothreitol (DTT, 100 mM) in PBS (1 mM EDTA, pH 8.0) for 2 h at room temperature, and then purified using Micro Bio-Spin P-6 Gel columns. The maleimide-activated proteins and reduced initiators (15-fold molar excess for IgGs; 7.5-fold for scFv, LaG-16-2; 3-fold for SpyCatcher) were mixed and reacted at room temperature for 2 h. HCR initiator-labeled proteins were purified using Amicon Ultra Centrifugal Filters (50 kDa MWCO) or Zeba spin columns (7000 MWCO).

[0040] For NHS-Azide conjugation, proteins were dialyzed into phosphate buffered saline (PBS, pH 7.4) and reacted with NHS-Azide (7.5-fold molar excess) at room temperature for 2 h. Excess crosslinkers were removed from azide-activated proteins using Zeba spin columns (7000 MWCO). The azide-activated proteins were mixed with DBCO-labeled HCR initiators (15-fold molar excess for IgGs; 7.5-fold for scFv, LaG-16-2; 3-fold for SpyCatcher) and then reacted at room temperature for 12 h. HCR initiator-labeled proteins were purified using Amicon Ultra Centrifugal Filters (50 kDa MWCO) or Zeba spin columns (7000 MWCO).

[0041] Cell culture and bacterial infections. HEK293T cells (ATCC CRL-3216) and HeLa cells (ATCC CCL-2) were used for the cultured-cell staining experiments. Cells were seeded on 12 mm #1.5 coverglass slips. Transfection was done using PEI. Cells were fixed with paraformaldehyde before subsequent experiments. The S. Typhimurium infection was performed according to a previous report.

[0042] Mice and virus injection. Animal care and use were in accordance with the institutional guidelines of the National Institute of Biological Sciences, Beijing (NIBS), as well as the governmental regulations of China.

[0043] Adult (8-12 weeks old) SERT-Cre mice [strain name: B6.Math.Cg-Tg(S1c6a4-Cre)ET33Gsat; MMRRC; Davis, Calif., USA], CaMKIIa-Cre [strain name: B6.Math.Cg-Tg(Camk2a-cre)T29-1Stl/J], ChAT-Cre [strain name: B6; 12956-Chattm2(cre)Lowl/J], and C57BL/6N mice of either sex were used. Mice were maintained with a 12/12 photoperiod (light on at 8 AM) and were provided food and water ad libitum. Mice were anaesthetized with pentobarbital (i.p., 80 mg×kg.sup.−1) before surgery, and then placed in a mouse stereotaxic instrument. For each mouse, 350 nL of virus (AAV-DIO-mGFP, AAV-DIO-mSNAPf, or AAV-DIO-4×SNAPf) was infused into the target areas of mice via a glass pipette at rate of 50 nL.Math.min.sup.−1. All subsequent experiments were performed at least 3 weeks after virus injection to allow sufficient time for transgene expression.

[0044] Tissue sample preparation. Mice were anesthetized with an overdose of pentobarbital and perfused intracardially with PBS, followed by paraformaldehyde (PFA, 4% wt/vol in PBS). Tissues were dissected out and postfixed in 4% PFA for 4 h at room temperature or 1 d at 4° C. Tissue samples were first dehydrated in 30% sucrose solution for preparing thin sections (50 μm). Thin sections were prepared on a Cryostat microtome (Leica CM1950).

[0045] Immunohistochemistry. The detailed information, working concentrations, and incubation times for antibodies can be found in Table 2. For brain sections and cultured cells, samples were permeabilized with 0.3% Triton X-100 in PBS (PBST) and blocked in 2% BSA in PBST at room temperature for 1 h. Sections were then incubated with primary antibodies. Samples were washed three times in PBST and were then incubated with biotinylated or HCR initiator-conjugated secondary antibodies. For control experiments, we used a mixture containing equal amounts of fluorophore-conjugated secondary antibodies and biotinylated secondary antibodies. Samples were then washed again three times in PBST. The biotinylated secondary antibodies were visualized by fluorophore-conjugated Streptavidin or DNA-fluorophore HCR amplifiers. HCR initiator-conjugated secondary antibodies were visualized by DNA-fluorophore HCR amplifiers.

[0046] Labeling of isHCR initiators. 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 ddH.sub.2O]. After labeling with biotinylated secondary antibodies, samples were incubated in 1 μg.Math.mL.sup.−1 streptavidin at room temperature for 30 min. After being washed three times in PBST, samples were incubated with 0.5 μM DNA-biotin HCR initiators at room temperature for 30 min. Samples were then washed three times and stored in PBST.

[0047] For multiplexed amplification using multiple biotinylated secondary antibodies (FIG. 1a), the immunosignals of target proteins were amplified using isHCR sequentially. That is, after being labeled with two primary antibodies, samples were incubated with one of two biotinylated secondary antibodies against a primary antibody; the basic isHCR amplification protocol was then used to amplify the signal of the secondary antibody. Next, before the application of the second of the two biotinylated secondary antibodies, brain sections were incubated with streptavidin (0.5 μg.Math.mL.sup.−1, 30 min at room temperature) to block any unbound biotin units remaining on the first secondary antibody; biotin (5 ng.Math.mL.sup.−1, 30 min at room temperature) was then added to saturate the biotin binding sites of the streptavidin. Having blocked the reactivity of the first biotinylated secondary antibody, the second biotinylated secondary antibody was added and then amplified. For multiplexed labeling using HCR initiator-conjugated secondary antibodies (FIGS. 2c, 2d), the snap-cooled DNA-fluorophore HCR amplifiers are applied directly to initiator-labeled samples and then amplified with the basic isHCR amplification protocol (i.e., lacking any streptavidin step).

[0048] The labeling of genetically encoded tags (SNAP-tag, SpyTag, GFP, and smFP_GCN4) with HCR initiators was conducted as follows (FIG. 2e and FIG. 1b-1d). After membrane permeabilization, cultured-cell or brain-section samples were incubated with appropriate binding partners. For SNAP-tag labeling, we applied 0.1 μM BG-labeled HCR initiators or 0.5-1 μM SNAP-Surface Alexa Fluor 546 and incubated these samples at room temperature for 1 h. For SpyTag labeling, we applied 25 μM HCR initiator-labeled SpyCatcher and incubated these samples at room temperature for 2 h. For mGFP-labeled samples, we applied 1 μg.Math.mL.sup.−1 HCR initiator-labeled LaG-16-2 and incubated these samples overnight at 4° C. For smFP_GCN4 labeling, we applied 5 μg.Math.m.sup.−1 HCR initiator-labeled scFv-GCN4-HA-GB1 and incubated these samples at room temperature for 1 h. PBS was used as incubation buffer for SNAP-Surface Alexa Fluor 546. HCR amplification buffer was used for all HCR initiator-containing reagents. Samples were then washed three times with PBST, and stored in PBST.

[0049] isHCR amplification. Note that while the experimental steps regarding the isHCR initiators varied according the conjugation strategies, the basic isHCR amplification process is common to all of the experiments. First, HCR amplification buffer was prepared [5×sodium chloride citrate (SCC buffer), 0.1% vol/vol Tween-20, and 10% wt/vol dextran sulfate in ddH.sub.2O]. Next, a pair of DNA-fluorophore HCR amplifiers were snap-cooled separately in 5×SSC buffer by heating at 95° C. for 90 s and cooling to room temperature over 30 min. Both of these amplifiers were then added to amplification buffer (typically to a final concentration of 12.5 nM for thin sections, or 150 nM for large volume samples). isHCR amplification proceeded as samples were incubated with this buffer overnight at room temperature, and free amplifiers were then removed by washing the three times with PBST prior to signal detection. Note that an additional graphene oxide step was added to this basic process for applications that demands background suppression. Briefly, to include the quenching step, GO (20 μg.Math.mL.sup.−1) was mixed with the amplifiers in amplification buffer. The amplifier/GO mixture was vortexed thoroughly and incubated at room temperature for at least 5 min before being added to initiator-labeled samples.

[0050] To perform multi-round amplification, we used DNA-biotin HCR amplifiers. Before use, DNA-biotin HCR amplifiers were snap-cooled. Samples were incubated with 12.5 nM DNA-biotin HCR amplifiers overnight at room temperature. After extensive washing, streptavidin (1 μg.Math.mL.sup.−1) was applied again to start the next round of amplification. The procedure of adding DNA-biotin HCR amplifiers and then streptavidin was repeated two or three times to achieve desired signal intensity. DNA-fluorophore amplifiers (12.5 nM) were used in the final round to visualize the signals. For control experiments, biotin and Alexa Fluor-488 dual-labeled HCR amplifiers were used for the first round of amplification. Alexa Fluor-546-labeled HCR amplifiers were used for the second round of amplification.

[0051] Fluorescence microscopy. Confocal microscopy was performed on a Zeiss Meta LSM510 confocal scanning microscope using a 10×0.3 NA, a 20×0.5 NA, a 63×1.4 NA, or a 100×1.3 NA objective, or on a Zeiss LSM880 confocal scanning microscope using a 20×0.5 NA or a 40×0.75 NA objective. Images were processed and measured with FIJI and Matlab.

[0052] To image the entire brain sections, we performed wide-field fluoresce imaging using the Olympus VS120 virtual microscopy slide scanning system with a 10× objective. For slide scanner imaging, brain sections from both groups on the same slide were imaged during the same imaging run using identical light intensity and exposure time. The images were acquired at 16 bit and were converted directly to the TIFF format for publication.

[0053] Statistical significance was determined using t-test or Kolmogorov-Smirnov test. P<0.05 was considered significant.

TABLE-US-00001 TABLE 1 Oligo nucleotide sequences and modifications Name Sequence (5′ to 3′) Modifications B1 I2 ATATAgCATTCTTTCTTgAggAgggCAg 5′ Biotin CAAACgggAAgAg (SEQ ID NO: 1) B1 I2 Amine ATATAgCATTCTTTCTTgAggAgggCAg 5′ Amine CAAACgggAAgAg (SEQ ID NO: 1) B1 I2 Thiol ATATAgCATTCTTTCTTgAggAgggCAg 5′ Thiol CAAACgggAAgAg (SEQ ID NO: 1) B1 I2 DBCO ATATAgCATTCTTTCTTgAggAgggCAg 5′ DBCO CAAACgggAAgAg (SEQ ID NO: 1) B1 Amplifier H1 546 CgTAAAggAAgACTCTTCCCgTTTgCTg 5′ Alexa Fluor CCCTCCTCgCATTCTTTCTTgAggAggg 546 CAgCAAACgggAAgAg  (SEQ ID NO: 2) B1 Amplifier H2 546 gAggAgggCAgCAAACgggAAgAgTCTT 3′ Alexa Fluor CCTTTACgCTCTTCCCgTTTgCTgCCCT 546 CCTCAAgAAAgAATgC  (SEQ ID NO: 3) B1 Amplifier H1 CgTAAAggAAgACTCTTCCCgTTTgCTg 5′ Biotin Terminal Biotin CCCTCCTCgCATTCTTTCTTgAggAggg CAgCAAACgggAAgAg  (SEQ ID NO: 2) B1 Amplifier H2 gAggAgggCAgCAAACgggAAgAgTCTT 3′ Biotin Terminal Biotin CCTTTACgCTCTTCCCgTTTgCTgCCCT CCTCAAgAAAgAATgC  (SEQ ID NO: 3) B1 Amplifier H1 CgTAAAggAAgACTCTTCCCgTTTgCTg Internal Biotin Internal Biotin CCCTCCTCgCATTCTTTCTTgAggAggg CAgCAAACgggAAgAg  (SEQ ID NO: 2) B1 Amplifier H2 gAggAgggCAgCAAACgggAAgAgTCTT Internal Biotin Internal Biotin CCTTTACgCTCTTCCCgTTTgCTgCCCT CCTCAAgAAAgAATgC  (SEQ ID NO: 3) B1 Amplifier H1 CgTAAAggAAgACTCTTCCCgTTTgCTg Internal Biotin Internal Biotin CCCTCCTCgCATTCTTTCTTgAggAggg 5′ Alexa Fluor and 5′-488 CAgCAAACgggAAgAg  488 (SEQ ID NO: 2) B1 Amplifier H2 gAggAgggCAgCAAACgggAAgAgTCTT Internal Biotin Internal Biotin CCTTTACgCTCTTCCCgTTTgCTgCCCT 3′ Alexa Fluor and 3′-488 CCTCAAgAAAgAATgC  488 (SEQ ID NO: 3) B5 I2 ATATACACTTCATATCACTCACTCCCAA 5′ Biotin TCTCTATCTACCC (SEQ ID NO: 4) B5 I2 Thiol ATATACACTTCATATCACTCACTCCCAA 5′ Thiol TCTCTATCTACCC (SEQ ID NO: 4) B5 I2 DBCO ATATACACTTCATATCACTCACTCCCAA 5′ DBCO TCTCTATCTACCC (SEQ ID NO: 4) B5 Amplifier H1 488 ATTggATTTgTAgggTAgATAgAgATTg 5′ Alexa Fluor ggAgTgAgCACTTCATATCACTCACTCC 488 CAATCTCTATCTACCC  (SEQ ID NO: 5) B5 Amplifier H2 488 CTCACTCCCAATCTCTATCTACCCTACA 3′ Alexa Fluor AATCCAATgggTAgATAgAgATTgggAg 488 TgAgTgATATgAAgTg  (SEQ ID NO: 6) B4 I2 Thiol ATATACACATTTACAGACCTCAACCTAC 5′ Thiol CTCCAACTCTCAC (SEQ ID NO: 4) B4 Amplifier H1 647 gAAgCgAATATggTgAgAgTTggAggTA 5′ Alexa Fluor ggTTgAggCACATTTACAgACCTCAACC 647 TACCTCCAACTCTCAC  (SEQ ID NO: 7) B4 Amplifier H2 647 CCTCAACCTACCTCCAACTCTCACCATA 3′ Alexa Fluor TTCgCTTCgTgAgAgTTggAggTAggTT 647 gAggTCTgTAAATgTg  (SEQ ID NO: 8)

TABLE-US-00002 TABLE 2 Antibodies Primary antibodies: Epitope Vendor Cat. No. Dilution Incubation Time Tyrosine Hydroxylase (TH) Millipore ab152 1:1000 Overnight at 4° C. for brain sections Choline acetyltransferase Millipore AB144P 1:500 Overnight at 4° C. for (ChAT) brain sections DOPA decarboxylase Abeam ab3905 1:500 24 h at 4° C. for brain (AADC) sections Neuronal nitric oxide Sigma N7280 1:500 Overnight at 4° C. for synthase (nNOS) brain sections Dopamine Transporter Millipore MAB369 1:500 Overnight at 4° C. for (DAT) brain sections hGBP1 Santa Cruz sc-53857 1:1000 1 h at RT for Western blot GFP Thermo Fisher A10259 1:1000 Overnight at 4° C. for Scientific brain sections; 1 h at RT for cultured cells and western blotting Ki67 eBioscience 14-5698- 1:1000 1 h at RT for cultured 80 cells Tom20 Santa Cruz sc-11415 1:1000 1 h at RT for cultured cells HA BioLegend 901505 1:500 1 h at RT for Western blot Secondary antibodies: Secondary ab. Vendor Label Cat. No. Dilution Incubation Time Goat anti- Abcam Biotin ab6720 1:1000 2 h at RT for brain rabbit sections Donkey anti- Jackson Biotin 705-065- 1:1000 2 h at RT for brain goat ImmunoResearch 147 sections Donkey anti- Jackson Biotin 715-065- 1:1000 1 h at RT for mouse ImmunoResearch 151 5 cultured cells Goat anti- Thermo Fisher DNA 31212 μg .Math. 2 h at RT for brain rabbit scientific HCR mL.sup.−1 sections; 1 h at RT initiators for Western blot and cultured cells Goat anti-rat Thermo Fisher DNA 31220 5 2 h at RT for brain scientific HCR μg .Math. sections; 1 h at RT initiators mL.sup.−1 for Western blot and cultured cells Fluorescent reagents: Incubation Name Vendor Label Cat. No. Conc. Time SNAP-Surface NEB Alexa Fluor S9132S 500 μM, 30 min at RT Alexa Fluor 546 546 1:500- 1:1000

TABLE-US-00003 TABLE 3 The protein sequence of smFP_GCN4. A total of nine GCN4 tags are inserted into a superfolder GFP scaffold. MEELLSKNYHLENEVARLKKGSGSGEELLSKNYHLENEVARLKKGS GSGEELLSKNYHLENEVARLKKGSGSGSKGEELFTGVVPILVELDG DVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLGG GVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEV KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQK NGIKANFKIRHNVEGSGSGEELLSKNYHLENEVARLKKGSGSGEEL LSKNYHLENEVARLKKGSGSGEELLSKNYHLENEVARLKKGSGSGD GSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMV LLEFVTAAGITHGMDELYKGSGSGEELLSKNYHLENEVARLKKGSG SGEELLSKNYHLENEVARLKKGSGSGEELLSKNYHLENEVARLKK (SEQ ID NO: 9)

Example 1

[0054] Multiplexed Labeling Using isHCR.

[0055] FIG. 1. (a) shows images of the dorsal striatum in mouse brain sections double immunostained for TH (green) and choline acetyltransferase (ChAT, red). The signals of each antigen were visualized sequentially using corresponding biotinylated secondary antibodies and isHCR. (b) HCR initiators were conjugated to GFP-nanobodies (LaG-16-2) using SMCC as the linker. mGFP proteins were expressed in the orbitofrontal cortex neurons in CaMKII-Cre transgenic mice using adeno-associated virus (AAV) vectors. The GFP signals in the superior colliculus were amplified using HCR initiator-conjugated GFP-nanobody and isHCR-546. (c) Schematic of rapid labeling using genetically encoded protein tags. Functionalized HCR initiators bind directly to protein tags and initiate the amplification process. AAV vectors that bear the Cre-dependent double-floxed inverse (DIO) open reading frame cassette containing genes encoding the SNAPf tag were constructed and packaged into AAV particles. The AAV vectors were injected into Cre-transgenic mice to achieve cell-type specific expression. (d) Confocal images of HEK293T cells and mouse brain labeled by SNAP-tag. The upper panel shows cells transiently expressing the mSNAPf-tag. The middle panel shows the DRN from SERT-Cre mice injected with AAV-DIO-mSNAPf. The bottom panel shows the medial septum from SERT-Cre mice injected with AAV-DIO-4×SNAPf. The tag-positive cells were labeled with benzylguanine-conjugated Alexa Fluor 546 (BG-546) or isHCR-546. Scale bars, 50 μm (a), 200 μm (b), 100 μm (d).

Example 2

[0056] Simultaneous Detection of Multiple Targets Using isHCR.

[0057] FIG. 2. (a) shows that two orthogonal HCR initiators can be conjugated directly onto secondary antibodies using either SMCC or Click Chemistry groups as linkers. (b) Western blot of a protein mixture containing purified hGBP1 and purified GFP-hGBP1 proteins. An anti-GFP primary antibody and an anti-hGBP1 primary antibody were applied. The two primary antibodies were detected using two secondary antibodies that were conjugated with different HCR initiators. (c) Images of HEK 293T cells immunostained against the nuclear protein Ki67 (red) and a mitochondria protein Tom20 (green) using two HCR initiator-conjugated secondary antibodies. The signals were then simultaneously amplified using isHCR-546 for Ki67 and isHCR-488 for Tom20. (d) Images of the dorsal striatum in mouse brain sections double immunostained against dopamine transporter (DAT, red) and neuronal nitric oxide synthase (nNOS, green) using two HCR initiator-conjugated secondary antibodies. The signals were then amplified simultaneously using isHCR-546 for DAT and isHCR-488 for nNOS. (e) Images of HEK 293T cells expressing three orthogonal protein tags targeting different cellular locations. The signals were simultaneously amplified using isHCR-488 for msmFP_GCN4, isHCR-546 for mitoSNAP, and isHCR-647 for H2B-SpyTag. Scale bar, 10 μm (c, e), 50 μm (d).