WO2019144391A1

WO2019144391A1 - Use of ishcr for exm and solvent-based tissue clearing

Info

Publication number: WO2019144391A1
Application number: PCT/CN2018/074365
Authority: WO
Inventors: Rui Lin; Minmin LUO
Original assignee: National Institute of Biological Sciences Beijin
Current assignee: National Institute of Biological Sciences Beijin
Priority date: 2018-01-26
Filing date: 2018-01-26
Publication date: 2019-08-01
Anticipated expiration: 2020-07-26

Abstract

A method for optimizing isHCR for ExM, and an optimized isHCR for 3DISCO-based tissue-clearing method are provided.

Description

Use of isHCR for ExM and solvent-based tissue clearing

Background

Owing to their ease of use, speed, and cost effectiveness, antibody-based immunoassays remain the most popular methods for detecting and identifying the location of proteins and other biomolecules in biological samples. These methods use a primary antibody that binds selectively to a target molecule (antigen) , and this antibody-antigen interaction can be visualized via a conjugated reporter or a labeled secondary antibody that can recognize and react with the primary antibody-epitope complex (Han, K.N., Li, C.A. &Seong, G.H. Annu. Rev. Anal. Chem. 6, 119-141 (2013) ) . 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 such as horseradish peroxidase (HRP) and alkaline phosphatase, which catalyze the deposition of chromogenic substrates on target complexes (Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. &Litt, G.J. J. Immunol. Methods 125, 279-285 (1989) ) . Fluorogenic substrates, especially those based on HRP-tyramide reaction chemistries, have been developed to support high-resolution fluorescence microscopy ³. Although very useful and widely-employed, current amplification methods have several drawbacks: they often generate high background, they can reduce spatial resolution due to dye diffusion, they are difficult to use for the simultaneous detection of multiple amplified signals (Carvajal-Hausdorf, D.E., Schalper, K.A., Neumeister, V.M. &Rimm, D.L. Lab. Invest. 95, 385-396 (2015) ) , and they are unsuitable for use with large-volume samples in several powerful new tissue expansion and clearing techniques.

We speculated that an enzyme-free amplification approach could overcome many of these limitations. We pursued this idea by attempting to adapt hybridization chain reaction (HCR) technology to amplify immunosignals ^10-12. HCR, which is based on recognition and hybridization events that occur between sets of DNA hairpin oligomers that self-assemble into polymers, has to date been used primarily for the amplification of mRNA signals from in situ hybridization samples (Choi, H.M.T., Beck, V.A. &Pierce, N.A. ACS Nano 8, 4284-4294 (2014) ; Shah, S. et al. Development 143, 2862-2867 (2016) ) , and more recently for the detection of protein- protein interactions (Koos, B. et al. Nat. Commun. 6, 7294 (2015) ) . In a typical usage case, nucleic acid probes complementary to the target mRNA molecule are used as ‘initiator’ oligos. Starting from the initiator oligos, a series of polymerization reactions are used to add fluorophore-labeled nucleic acid ‘amplifier’ oligos to the target mRNA-initiator complex; the fluorophores are then visualized.

Recent developments in tissue preparation techniques are revolutionizing the high-resolution imaging of very large-volume samples. For example, expansion microscopy (ExM) achieves super-resolution imaging by expanding a sample tissue in a water-swellable expansion polymer gel. However, since the expansion process increases the overall sample volume by at least 2-3 orders of magnitude, ExM inevitably results in a dramatic dilution of any experimental reagents (e.g., fluorescent probes for immunosignal detection) ; indeed, the developers of ExM have emphasized a pressing need for a signal amplification technology that is compatible with ExM.

Another exciting trend in the field of biological imaging is the emergence of tissue clearing methods that can be used with large-volume samples. These methods, such as the recently-developed uDISCO, can render thick tissues (e.g., whole organs) transparent, allowing for rapid fluorescence microscopy analysis at subcellular resolution.

Summary of the Invention

The invention provides a method for optimizing isHCR for ExM, which combines a binder-biomolecule interaction with Hybridization Chain Reaction (HCR) for amplifying immunosignals, and simultaneously, the initiators used in the isHCR are functionalized to both bind to the target and anchor itself to the swellable polymer during gelation process of ExM. The invention further provides an optimized isHCR for 3DISCO-based tissue-clearing methods, such as uDISCO and iDISCO, which comprises an additional round of fixation with formaldehyde to crosslink the HCR initiator-amplifier polymers with nearby proteins before clearing, wherein the isHCR combines a binder-biomolecule interaction with hybridization Chain Reaction (HCR) for amplifying immunosignals.

In one aspect, the invention provides a method for optimizing isHCR for ExM, which combines a binder-biomolecule interaction with Hybridization Chain Reaction (HCR) , wherein the initiator is functionalized to anchor themselves to the swellable polymer during gelation process of ExM.

The initiator can be functionalized with moieties, such as acrydite, amine, acrylamide, 6- ( (acryloyl) amino) hexanoic acid or methacrylic acid for anchoring the swellable polymer during gelation process of ExM, and the initiator is also bound to an antibody. Following the gelation of ExM, a pair of DNA-fluorophore HCR amplifiers is applied.

The labeling clusters that result from the conventional enzyme-based amplification, as in tyramide amplification, are attached to the adjacent proteins of the target molecule. The labeling clusters will inevitably be destroyed during the protein digestion step of ExM. Even if the enzyme-based labeling clusters manage to survive the protein digestion, the individual clusters will inevitably expand together with the whole sample, thereby severely decreasing the labeling resolution and sensitivity.

In contrast, with the use of isHCR for ExM applications, only HCR initiators are incorporated into the expansion gel during the gelation process. HCR amplifiers are added after gelation and are not directly linked with the gel. Therefore, the individual fluorescent amplification polymers themselves will not expand during the final expansion step. This is the key advantage of isHCR over conventional enzyme-based amplification methods for ExM applications.

Specifically, this approach produced strong and high-resolution Vglut3 labeling in puncta, whereas combining standard immunostaining methods with ExM generated much weaker signals and many fewer labeled puncta -a greater than 4000-fold increase in the signal intensity was achieved by isHCR amplification.

The HCR initiators can be hybridized with any of several types of self-assembling DNA HCR amplifiers, including a fluorophore-labeled oligo that can be used for visualization of the original target signal.

HCR initiator can be conjugated to an antibody using many interactions, such as the streptavidin-biotin, covalent bonds (chemical linkers, e.g., amine-reactive linkers or click chemistry linkers) , and etc. The amine-reactive linkers can be linkers that contain the succinimidyl ester group. The click chemistry linkers can be linkers that contain the click chemistry functional groups.

Preferably, the HCR initiator and amplifier (H1 and/or H2) used in the present isHCR method can be terminally modified or internally modified for improving the signal strength or as an interface to access other chemical reactions. The HCR initiator and amplifier (H1 and/or H2) used in the present isHCR method can be terminally modified or internally modified with chemical linkers and/or fluorescent dyes. For example, the HCR initiator and amplifiers (H1 and/or H2) used in the present isHCR method can be terminally modified or internally modified with biotin, acrydite, amine, thiol, digoxigenin, DBCO, TCO, Tetrazine, Alkyne, FITC, Cyanine dyes, Alexa Fluors, Dylight fluors, Atto dyes or Janelia Fluor dyes, whererin the Alexa Fluors is Alexa Fluro 546, Alexa Fluor 488, or Alexa Fluor 647.

Preferably, the isHCR method uses biotin-streptavidin interaction, wherein DNA-biotin HCR initiator is attached to a biotinylated antibody and in turn trigger the self-assembly of DNA-fluorophore HCR amplifiers into fluorescent polymers.

The isHCR method uses label-free streptavidin, which allows the attachment of synthesized 5’-biotinylated DNA HCR initiators to the vacant binding sites of streptavidin, which is attached to the biotinylated antibody.

The biotinylated antibody can be a biotinylated secondary antibody that reacts with a primary antibody specific to a target analyte. The secondary antibody is a IgG or a Nanobody, and the primary antibody is a IgG, a Nanobody or a scFv.

The isHCR may be multi-round isHC, in which an amplifier or a pair of amplifiers are modified to access branched multiple-round amplification in order to branch and grow the HCR polymers.

The isHCR can also be optimized for multiplexed labeling, wherein orthogonal binders for conjugating orthogonal initiators and targeting multiple target biomolecules, and orthogonal initiators directed to orthogonal binders respectively are used in HCR to allow HCR amplification of multiple target biomolecules.

In this situation, the binder can be an antibody, a fragment of an antibody, or a genetically-engineered protein tag. If the orthogonal binders are orthogonal antibodies, the antibodies may be biotinylated antibodies, and the orthogonal HCR initiators may be biotinylated initiators for conjugating the vacant binding sites of streptavidin, which is capable of conjugating to the biotinylated antibodies in order to sequentially amplify multiple target biomolecules.

Preferably, the orthogonal HCR initiators may be directly conjugated to the orthogonal antibodies using chemical linkers so as to simplify the multiplexed labeling procedure. The chemical linkers can be amine-reactive linkers, thiol-reactive linkers or click chemistry groups. For example, the orthogonal HCR initiators can be conjugated directly onto the antibodies via SMCC or NHS-Azide linkers. This direct conjugation allows simultaneous HCR amplification directed to multiple target biomolecules.

Preferably, the antibody may be a secondary antibody that reacts with a primary antibody specific to an analyte, the secondary antibody may be a IgG or a Nanobody, and the primary antibody may be a IgG, aNanobody or a scFv.

In the situation that the binder is a genetically-engineered protein tag, the orthogonal HCR initiators can be conjugated to tag binding partners, which are capable of binding tags labeling different target biomolecules. The biomolecules can be biomolecules, such as proteins, small signaling molecules, neurotransmitters, etc., in the cells. The tags have the chemical groups that are nonreactive toward the biomolecules, such as amines or carboxyl moieties. The HCR initiators are conjugated to tag binding partners, and subsequently are used for HCR amplification to detect tags. The persons skilled in the art may easily choose the tags and tag binding partners as desired.

The tags may be orthogonal tags targeting different cellular locations and being expressed in cultured cells. In this situations, the HCR initiators may be conjugated to tag binding partners (for example, SpyCatcher, SnoopCatcher, benzylguanine (BG) , and scFv respectively) , and subsequently are used to detect the subcellular localization of the genetically-encoded tags (SpyTag, SnoopTag, SNAP-tag, and GCN4-tag respectively) . CLIP-tag and Halo-tag, two chemical tags that are orthogonal to the SNAP-tag technology, could also be adopted for HCR in a fashion similar to SNAP-tag. Recently, novel mini-protein binders that target small ligands were developed using de novo protein design. These new ligand-binder pairs, such as digoxigenin/DIG 10.3 also can be used with HCR.

The isHCR method can be used to powerfully amplify immunosignals at different subcellular locations (e.g., cell and vesicle membrane, cytosol, mitochondrion, and cell nucleus) and in various types of samples (e.g., blotting, cultured cells, tissue sections, and whole organ) .

The isHCR method will be especially useful in applications that require sensitive detection of immunosignals. We here demonstrated its use for amplifying immunosignals with monoclonal antibodies, for detecting the extremely low-abundance signal of translocated bacterial effectors, and for enhancing diluted immunosignals in ExM, among other applications. The actual amplification performance of isHCR depends on the particular applications and on the abundance of a given target signal: for signal amplification in western blotting and tissue section samples, isHCR outperformed standard IHC by up to 2 orders of magnitude; improvements of 3 orders of magnitude were achieved with ExM samples.

In another aspect, the invention further provides an optimized isHCR method for using in 3DISCO-based tissue-clearing methods, such as uDISCO and iDISCO, which comprises an additional round of fixation with formaldehyde to crosslink the HCR initiator-amplifier polymers with nearby proteins before tissue clearing, wherein the HCR combines a binder-biomolecule interaction with a Hybridization Chain Reaction (HCR) for amplifying immunosignals.

The isHCR comprises directly conjugating a HCR initiator oligo to an antibody (including but not limit to traditional IgGs and nanobodies) and hybridization chain reaction for amplifying immunosignals.

HCR initiators can be conjugated to antibodies using many interactions, such as the streptavidin-biotin, covalent bonds (chemical linkers, e.g., an amine-reactive linker, a thiol-reactive linker or a click chemistry linker) , and etc. The amine-reactive linkers can be linkers that contain the succinimidyl ester group. The click chemistry linkers can be linkers that contain the click chemistry functional groups. The click chemistry linker may be selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker.

Preferably, the HCR initiators and amplifiers (H1 and H2) used in the present isHCR method can be terminally modified or internally modified for improving the signal strength or as an interface to access other chemical reactions. The HCR initiators and amplifiers (H1 and H2) used in the present isHCR method can be terminally modified or internally modified with chemical linkers and/or fluorescent dyes. For example, the HCR initiators and amplifiers (H1 and H2) used in the present isHCR method can be terminally modified or internally modified with biotin, acrydite, amine, thiol, digoxigenin, DBCO, TCO, Tetrazine, Alkyne, FITC, Cyanine dyes, Alexa Fluors, Dylight fluors, Atto dyes or Janelia Fluor dyes, wherein the Alexa Fluor is Alexa Fluro 546, Alexa Fluor 488, or Alexa Fluor 647.

Preferably, the isHCR method uses biotin-streptavidin interaction, wherein DNA-biotin HCR initiator is attached to a biotinylated antibody, and in turn triggers the self-assembly of DNA-fluorophore HCR amplifiers into fluorescent polymers.

The biotinylated antibody can be a biotinylated secondary antibody that reacts with a primary antibody specific to a target analyte.

The present method is compatible with 3DISCO-based tissue-clearing methods, such as uDISCO and iDISCO. The optimized isHCR protocol allows isHCR components to survive the harsh clearing procedures used during 3DISCO-based tissue-clearing procedure, and results in much stronger labeling than standard IHC staining.

In addition, the present optimized isHCR method not only enabled immunosignal detection in the whole organ, for example whole adult lungs, but also achieved signal amplification evenly throughout the sample material.

The isHCR may be multi-round isHCR, in which an amplifier or a pair of amplifiers wherein the amplifier or the pair of amplifiers may be terminally modified or internally modified with a chemical group and/or a fluorescent dye, which allows initiating further rounds of amplification, the said chemical group is selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazine, Alkyne, and/or DBCO, and the said 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.

Preferably, a pair of fluorophore-tagged amplifiers are added to the final round of the multiple-round HCR for visualization.

Preferably, the amplifier or the pair of amplifiers may be modified at internal positions, which are accessible to streptavidins, which serve as anchors for each successive round of branching in multi-round HCR.

The isHCR can also be optimized for multiplexed labeling, wherein orthogonal binders for conjugating orthogonal intiators and targeting multiple target biomolecules, and orthogonal initiators directed to orthogonal binders respectively are used in HCR to allow HCR amplification of multiple target biomolecules.

Preferably, the antibody may be a secondary antibody that reacts with a primary antibody specific to an analyte, the secondary antibody is a IgG or a Nanobody, and the primary antibody is a IgG, a Nanobody or a scFv.

The invention encompasses all combination of the particular embodiments recited herein.

Brief Description of the Figures

Figure 1. Optimization of isHCR for expansion microscopy (ExM) and uDISCO-cleared brain tissue.

[Rectified under Rule 91, 12.03.2018]
Figure 2. Experimental steps for applying isHCR to various types of samples.
Figure 3. Expressed 4xSNAPf in the cholinergic interneurons in the striatum of the mouse brains.

Description of Particular Embodiments of the Invention

In the ExM tissue preparation process, all of the proteins in a sample are digested to facilitate isotropic expansion. Given that this digestion would destroy the original spatial information of a target immunosignal, it is clear that any meaningful application of isHCR to ExM would require some steps that preserved this spatial information about the target.

In the first embodiment, a type of HCR initiator capable of both binding to the target and anchoring itself to the swellable polymer is designed. To this end, the inventors functionalized DNA-biotin initiators with an acrydite moiety (for polymer anchoring) and then bound these new initiators to the antibody-biotin-streptavidin complex (Fig. 1a) . Following the gelation and protein digestion steps of ExM, DNA-fluorophore HCR amplifiers were applied. This approach produced strong and high-resolution Vglut3 labeling in puncta, whereas combining standard immunostaining methods with ExM generated much weaker signals and many fewer labeled puncta -a greater than 4000-fold increase in the signal intensity was achieved by isHCR amplification (Fig. 1b-d) . Importantly, no difference in the mean diameter of Vglut3-positive puncta was observed between the samples visualized with isHCR or standard IHC staining (Fig. 1d bottom) .

3DISCO-based tissue-clearing, such as the recently-developed uDISCO, can render thick tissues (e.g., whole organs) transparent, allowing for rapid fluorescence microscopy analysis at subcellular resolution.

The inventors conducted with brain sections indicated that isHCR performed worse than standard IHC in tissues cleared via uDISCO (Fig. 1e) , which indicated that organic solvents do not affect the antigen-antibody or antibody-streptavidin interactions but do disrupt the interactions among the HCR oligos.

In the second embodiment, before clearing, an additional round of fixation with formaldehyde to crosslink the HCR initiator-amplifier polymers with nearby proteins was applied. This simple fixation treatment allowed the isHCR components to survive the harsh clearing procedures used during uDISCO, and resulted in much stronger labeling than standard IHC staining (Fig. 1e) .

Specifically, the organic-solvent-based DISCO clearing methods (such as 3DISCO, iDISCO, iDISCO+, and uDISCO) achieve the highest level of transparency among all clearing approaches, and these methods require significantly shorter experimental times, and are in general easier to perform than aqueous solution-based clearing methods such as CLARITY and CUBIC. 3DISCO (uDISCO and iDISCO) requires the immunostaining to be performed prior to clearing, which requires the amplification complex to stay intact during the harsh clearing processes that use organic solvents. This is challenging, and has not been achieved by previous studies. In contrast, for those clearing methods that based on aqueous solutions (e.g., CLARITY or CUBIC) , the staining is performed after clearing. In fact, our isHCR should also be compatible with these aqueous solutions-based clearing methods.

In the third embodiment, the inventors next performed whole-mount immunostaining and isHCR amplification for Prosurfactant Protein C (proSP-C) in whole lungs sampled from adult mice using a DNA HCR initiator conjugated secondary antibody. After isHCR amplification and fixation, the lung samples were cleared using uDISCO (Fig. 1f) . Strikingly, isHCR not only enabled immunosignal detection in whole adult lungs (to our knowledge, the first instance of whole-mount immunostaining of adult lungs) , it was able to achieve signal amplification evenly throughout the sample material (cross-section images in the middle panel of Fig. 1f) .

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 (HI amplifer) , 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.

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.

Click chemistry is a class of biocompatib le rea ctions 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.

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

Expansion microscopy (ExM) is a process using a swellable polymer network within a specimen, which can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved.

3DISCO (Three-Dimensional Imaging of Solvent-Cleared Organs) is a process for clearing large tissues, which provides the ability to accurately and quickly acquire complete histological information about labeled cells/molecules in large tissues. This process consists of sequential solution incubation steps. 3DISCO based tissue-clearing method involves for example, uDISCO, iDISCO and iDISCO+. Ultimate DISCO (uDISCO) is a process for clearing bigger samples, such as tissues or whole organs. The clearing consisted of serial incubations of the fixed samples in 5-80ml of 30vol%, 50vol%, 70vol%, 80vol%, 90vol%, 96vol%and 100vol%tert-butanol at 34-35℃ to dehydrate the tissue, followed by immersion in DCM for 45-60 min at room temperature to remove the lipids. Eventually, they were incubated in BABB or DiBenzyl Ether at room temperature for at least 2 hours until samples became transparent.

Examples

Methods and Materials

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 ₂O and stored at -20℃.

The detailed information for antibodies and fluorescent reagents is shown in Supplementary Table 2. Methacrylic acid N-hydroxy succinimidyl ester (MA-NHS, 730300) was obtained from Sigma-Aldrich and was dissolved in anhydrous DMSO at a concentration of 1 M and stored at -20℃ until use. 4-hydroxy-TEMPO (4-HT, 176141) , Ammonium persulfate (APS, A3678) , Tetramethylethylenediamine (TEMED, T7024) , sodium acrylate (408220) , DiBenzyl Ether (DBE, 108014) , and Dextran sulfate (D8906) were purchased from Sigma-Aldrich. Tert-Butanol (974855) was purchased from J&K. Proteinase K (25530049) was purchased from Thermo Fisher Scientific. Graphene Oxide (GO, XF020, particle size ＜ 500 nm, C/O ratio = 1.6) was obtained from Nanjing XFNANO.

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 (50kDa MWCO) or Zeba spin columns (7000 MWCO) .

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 12h. HCR initiator-labeled proteins were purified using Amicon Ultra Centrifugal Filters (50kDa MWCO) or Zeba spin columns (7000 MWCO) .

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.

Adult (8-12 weeks old) SERT-Cre mice [strain name: B6. Cg-Tg (Slc6a4-Cre) ET33Gsat; MMRRC; Davis, CA, USA] , and C57BL/6N mice of either sex were used. Mice were maintained with a 12/12 photoperiod (light on at 8AM) and were provided food and water ad libitum. Mice were anaesthetized with pentobarbital (i.p., 80 mg×kg ^-1) .

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 (brains or lungs) were dissected out and postfixed in 4%PFA for 4 h at room temperature or 1 d at 4℃. Tissue samples were first dehydrated in 30%sucrose solution for preparing thin sections (50 μm) or, for large volume tissue samples (lungs and brain sections thicker than 500 μm) , pretreated with methanol according to the original iDISCO+ protocol. The thickness of mouse brain sections was 50 μm for immunofluorescent labeling or for expansion microscopy (ExM) . Thin sections were prepared on a Cryostat microtome (Leica CM1950) . For uDISCO-cleared brain samples, thick sections (500 μm) were prepared using a vibratome (Leica VT 1200S) . The brain section samples for those experiments that compared the signal intensity from isHCR amplification and traditional IHC methods were serial sections from the same mice, and were prepared on the same day. Serial sections were divided equally into two groups for the subsequent experiments.

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. For the samples that were stained by the standard IHC method using biotinylated secondary antibodies and subsequently prepared for expansion microscopy (ExM) analysis, sections were further incubated in 1 mM MA-NHS in PBS at room temperature for 1 h. 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.

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 ₂O] . After labeling with biotinylated secondary antibodies, samples were incubated in 1 μg·mL ^-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.

For multiplexed amplification using multiple biotinylated secondary antibodies, 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·mL ^-1, 30 min at room temperature) to block any unbound biotin units remaining on the first secondary antibody; biotin (5 ng·mL ^-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 (Fig. 3) , 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) .

The labeling of genetically encoded tags (SNAP-tag, SpyTag, GFP, and smFP_GCN4) with HCR initiators was conducted as follows (Fig. 3) . 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 1h. For SpyTag labeling, we applied 25 μM HCR initiator-labeled SpyCatcher and incubated these samples at room temperature for 2h. For mGFP-labeled samples, we applied 1 μg·mL ^-1 HCR initiator-labeled LaG-16-2 and incubated these samples overnight at 4℃. For smFP_GCN4 labeling, we applied 5 μg·mL ^-1 HCR initiator-labeled scFv-GCN4-HA-GB1 and incubated these samples at room temperature for lh. 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.

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 ₂O] . Next, a pair of DNA-fluorophore HCR amplifiers were snap-cooled separately in 5 ×SSC buffer by heating at 95℃ for 90s 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·mL ^-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.

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·mL ^-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.

Expansion microscopy (ExM) . The ExM protocols were performed following the original publications ^7, 40. In brief, 50 μm initiator-labeled sections were incubated for 45 min in gelling solution [1 × PBS, 2 M NaCl, 2.5% (wt/wt) acrylamide, 0.15% (wt/wt) N, N-ethylenebisacrylamide, 8.6% (wt/wt) sodium acrylate, 0.01% (wt/wt) 4-HT, 0.2% (wt/wt) TEMED, 0.2% (wt/wt) APS) ] at 4℃. Sections were then transferred to a gelling chamber and gelled at 37℃ for 2 h. Gels were incubated in digestion buffer (50 mM Tris pH 8.0, 1 mM EDTA, 0.5%Triton X-100, 0.8 M guanidine HCl, 8 units·mL ^-1 Proteinase K) overnight at room temperature. All samples were stored in PBS before subsequent isHCR amplification, isHCR amplification for ExM samples were performed as described above. Finally, gels were washed and expanded in ddH ₂O. The expanded gels were cut and mounted using ddH ₂O as the mounting medium. Gels were immobilized using epoxy to prevent drifting during microscopic imaging.

Tissue clearing. Tissue clearing was performed using a modified uDISCO protocol9, 27. Thick brain sections or lungs were immunostained and amplified by isHCR. The amplified sections were incubated in formaldehyde (4%) for 2h at room temperature. After washing three times in PBS, the sections were dehydrated via serial 2-hour incubations in 30%, 50%, 70%, 80%, 90%, 96%, and 100%tert-butanol (vol/vol in ddH ₂O) at 35℃. Finally, samples were incubated in DiBenzyl Ether (DBE) at room temperature until clear.

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. For confocal imaging, brain sections from both groups were imaged using identical laser intensity, pin hole value, detector gain, and offset. In some experiments, higher laser intensities were used to image brain sections for the SA group samples: such images have been labeled as ‘higher laser intensity’ in the figures.

Data analysis. To determine the width of immunopositive neuronal processes or cell membrane, we calculated the full width at half maximum (FWHM) using signals from either unamplified or isHCR-amplified channels. A series of straight lines perpendicular to the membrane or neuronal process were drawn. The corresponding intensity profiles were plotted and averaged for each channel. For neuronal process measurement, the baseline correction was applied. The corrected average intensity of each channel was fit to a Gaussian distribution with a non-linear least square method. FWHM was calculated with the equation:

where σ is the standard deviation of the fitted Gaussian curve. For cell membrane measurement, the mean intensity of the distance from the edge of the membrane or neuronal process (typically between -3 μm to -2 μm) was calculated as the baseline (denoted m) . The peak value of the curve was determined (denoted p) . Half-peak intensity (I) was defined (p+m) /2. The full width at half maximum (FWHM) was quantified as the width of the average intensity curve at I.

To quantify the size of Vglut3-immunopositive puncta, we first randomly chose immunopositive puncta using the image data from unamplified and isHCR-amplified samples. A straight line across each punctum was drawn and rotated using the punctum as the center of rotation. For every 6 degrees, the intensity profile along the line of both the unamplified and isHCR-amplified channel was plotted. The average intensity of each channel was calculated, and baseline correction was then applied. The FWHM was calculated using the same protocol for neuronal process measurement as described above.

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

Table 1. Oligo nucleotide sequences and modifications

Table 2. Antibodies

Primary antibodies:

Secondary antibodies:

Fluorescent reagents:

Example 1

Experimental steps for applying isHCR to various types of samples.

Please see Figure 2. Samples are first incubated with a given primary antibody and then its corresponding biotinylated secondary antibody. After washing, samples are incubated sequentially in streptavidin and DNA-biotin HCR initiators at room temperature (RT) . DNA-fluorophore HCR amplifiers are applied to visualize the signals. The amplifiers can be mixed with graphene oxide (GO) to reduce background fluorescence. If multiple rounds of amplification are desired, biotin-labeled HCR amplifiers are used, except for the final (visualization) round. For ExM samples, biotin and acrydite dual-labeled HCR initiators are used. HCR amplifiers are applied after gelation and digestion. For uDISCO-cleared samples, an additional formaldehyde fixation step is performed after HCR amplifier incubation. Fixed samples are then cleared according to the standard uDISCO protocol. If HCR initiator-conjugated secondary antibodies are used, the HCR amplifiers can be directly applied after wash. For samples that are labeled by genetically encoded tags, we apply initiator-conjugated binding partners and then HCR amplifiers after wash.

Example 2

Optimization of isHCR for expansion microscopy (ExM) and uDISCO-cleared brain tissue.

Figure 1 shows (a) A modified isHCR strategy for ExM. HCR initiators are dual-labeled with biotin at the 3’-end and acrydite at the 5’-end. The initiators bind to antibodies through the biotin group. During the gelation process, initiators are incorporated into gels through the acrydite group, and therefore survive the subsequent protein digestion. HCR amplifiers are applied after digestion. (b) Images of mouse brain sections immunostained against Vglut3 using isHCR and ExM. The upper panel shows the brain sections before (left) and after (right) expansion. The bottom panel shows the stack images of Vglut3-positive signals in VTA. (c) Images of the ventral tegmental area (VTA) in expanded brain sections immunostained for Vglut3 using isHCR-546 (left) or SA-546 (middle) . The rightmost panel shows a higher laser intensity image of SA-546. (d) isHCR dramatically increased the fluorescent signals (n = 3 replicates; 366 puncta from SA-546 samples and 424 puncta from isHCR-546 samples were analyzed; ****, P ＜ 0.001; t-test) . No difference of the mean diameter of Vglut3-positive puncta was observed (n = 3 replicates; 45 puncta from SA-546 samples and 100 puncta from isHCR-546 samples were analyzed; t-test) . (e) Images of uDISCO-cleared thick brain sections (500 μm) immunostained for TH using SA-546 or isHCR546. Crosslinking HCR amplification polymers with adjacent proteins by formaldehyde fixation preserved the amplified fluorescence signals after uDISCO clearing. (f) The lung of an adult mouse was immunostained against Prosurfactant Protein C (proSP-C) and cleared using uDISCO. The middle panel shows the cross-section images of the two sections indicated in the left panel. The right panel shows a zoomed-in image of the boxed region in the middle panel. Scale bars, 3 mm (b upper) , 10 μm (b lower and c, adjusted according to an expansion factor of 4) , 50 μm (e, f right panel) , 1 mm (f left panel) , 500 μm (f middle panel) .

Example 3

To further test the applicability of isHCR in thick brain tissue samples, we performed SNAP-tag labeling and isHCR amplification in thick mouse brain slices. We selectively expressed 4xSNAPf in the cholinergic interneurons in the striatum of the mouse brains (Fig. 3, inlet in the left panel) . We prepared 1-mm brain slices and linked SNAP tags directly with BG-functionalized HCR initiators. The signals were then visualized using isHCR. Strong signals were observed throughout the striatum (Fig. 3) , demonstrating that isHCR can be applied with chemical tags to achieve antibody-independent labeling of thick tissue samples.

Claims

A method for optimizing Hybridization Chain Reaction (HCR) for ExM, which combines a binder-biomolecule interaction with a Hybridization Chain Reaction (HCR) , wherein the initiator in the HCR is functionalized to anchor themselves to a swellable polymer during gelation process of ExM.
The method of claim 1, wherein the initiator is functionalized with a moiety selected from acrydite, amine, acrylamide, 6- ( (acryloyl) amino) hexanoic acid and methacrylic acid.
The method of claim 1 or 2, wherein a pair of HCR amplifiers are applied after the gelation of ExM.
The method of claim any one of claims 1-3, wherein the binder is an antibody, a fragment of an antibody, or a genetically-engineered protein tag.
The method of any one of claims 1-4, wherein the HCR initiator has a region for hybridizing with a HCR amplifier, and a region for conjugating an antibody specific to an analyte.
The method of any one of claims 1-5, wherein the HCR initiator and/or the amplifier are terminally modified and/or internally modified with biotin, acrydite, amine, thiol, DBCO, and/or fluorescent dye, and said 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.
The method of any one of claims 1-6, wherein the HCR initiator is conjugated to an antibody via an interaction selected from streptavidin-biotin interaction and covalent bond interaction.
The method of claim 7, wherein the HCR initiator is a biotinylated initiator, which is capable of attaching to the vacant binding sites of streptavidin, and the streptavidin is capable of attaching to a biotinylated antibody, and whereby the HCR initiator is conjugated to the antibody.
The method of claim 7, wherein the covalent bond interaction is an interaction via a chemical linker selected from an amine-reactive linker containing a succinimidyl ester group and a click chemistry linker.
The method of claim 9, wherein the click chemistry linker is selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker.
The method of any one of claims 1-10, wherein the amplifier or the pair of amplifiers are terminally modified or internally modified with a chemical group and/or a fluorescent dye, which allows initiating further rounds of amplification, the said chemical group is selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazine, Alkyne, and/or DBCO, and the said fluorescent dye is selected from FITC, Cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluro 546, Alexa Fluor 488, and Alexa Fluor 6477.
The method of claim 11, wherein a pair of fluorophore-tagged amplifiers are added to the final round of the multiple-round HCR for visualization.
The method of claim 11 or claim 12, wherein the amplifier or the pair of amplifiers are modified at internal positions, which are accessible to streptavidins, which serve as anchors for each successive round of branching in multi-round HCR.
The method of any one of claims 1-13, wherein orthogonal binders for conjugating orthogonal initiators and targeting multiple target biomolecules, and orthogonal initiators directed to orthogonal binders respectively are used in HCR to allow HCR amplification of multiple target biomolecules.
The method of claim 14, wherein the orthogonal HCR initiators are directly conjugated to the orthogonal antibodies using chemical linkers, said chemical linker is selected from an amine-reactive linker containing a succinimidyl ester group, a thiol-reactive linker or a click chemistry linker.
The method of claim 15, wherein the click chemistry linker is selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker.
The method of claim 15 or claim 16, wherein the antibody is a secondary antibody that reacts with a primary antibody specific to an analyte, the secondary antibody is a IgG or a Nanobody, and the primary antibody is a IgG, a Nanobody or a scFv.
The method of claim 14, wherein the orthogonal binders are genetically-engineered protein tags for labeling different target biomolecules, and the orthogonal HCR initiators are conjugated to tag binding partners respectively, which are capable of binding tags.
The method of claim 18, wherein the tag has a chemical group nonreactive toward a biomolecule, said chemical group is selected from an amine moiety, a carboxyl moiety, a thiol moiety and a glycosylated modification moiety.
The method of claim 18 or claim 19, wherein the HCR initiators are conjugated to tag binding partners selected from SpyCatcher, SnoopCatcher, benzylguanine (BG) , and scFv, and subsequently are used to detect the subcellular localization of the genetically-encoded tags selected from SpyTag, SnoopTag, SNAP-tag, and GCN4-tag respectively.
The method of claim 18 or claim 19, wherein the tag is CLIP-tag or Halo-tag.
A method for optimizing Hybridization Chain Reaction (HCR) for 3DISCO-based tissue-clearing methods, which comprises an additional round of fixation with formaldehyde to crosslink the HCR initiator-amplifier polymers with nearby proteins before tissue clearing of 3DISCO-based tissue-clearing methods, wherein the HCR combines a binder-biomolecule interaction with a Hybridization Chain Reaction (HCR) .
The method of claim 22, wherein the 3DISCO-based tissue-clearing method is selected from uDISCO, iDISCO and iDISCO+.
The method of claim 22 or claim 23, wherein the solvent used in 3DISCO-based tissue-clearing method is an organic solvent.
The method of claim 22 or claim 23, wherein the solvent used in 3DISCO-based tissue-clearing method is an aqueous solvent.
The method of claim any one of claims 22-25, wherein the binder is an antibody, a fragment of an antibody, or a genetically-engineered protein tag.
The method of any one of claims 22-26, wherein the HCR initiator has a region for hybridizing with a HCR amplifier, and a region for conjugating an antibody specific to an analyte.
The method of any one of claims 22-27, wherein the HCR initiator and/or the amplifier are terminally modified and/or internally modified with biotin, acrydite, amine, thiol, DBCO, and/or fluorescent dye, and said 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.
The method of any one of claims 22-28, wherein the HCR initiator is conjugated to an antibody via an interaction selected from streptavidin-biotin interaction and covalent bond interaction.
The method of claim 29, wherein the HCR initiator is a biotinylated initiator, which is capable of attaching to the vacant binding sites of streptavidin, and the streptavidin is capable of attaching to a biotinylated antibody, and whereby the HCR initiator is conjugated to the antibody.
The method of claim 30, wherein the covalent bond interaction is an interaction via a chemical linker selected from an amine-reactive linker containing a succinimidyl ester group and a click chemistry linker.
The method of claim 31, wherein the click chemistry linker is selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker.
The method of any one of claims 22-32, wherein the amplifier or the pair of amplifiers are terminally modified or internally modified with a chemical group and/or a fluorescent dye, which allows initiating further rounds of amplification, the said chemical group is selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazine, Alkyne, and/or DBCO, and the said fluorescent dye is selected from FITC, Cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluro 546, Alexa Fluor 488, and Alexa Fluor 6477.
The method of claim 33, wherein a pair of fluorophore-tagged amplifiers are added to the final round of the multiple-round HCR for visualization.
The method of claim 33 or claim 34, wherein the amplifier or the pair of amplifiers are modified at internal positions, which are accessible to streptavidins and which serve as anchors for each successive round of branching in multi-round HCR.
The method of any one of claims 22-35, wherein orthogonal binders for conjugating orthogonal initiators and targeting multiple target biomolecules, and orthogonal initiators directed to orthogonal binders respectively are used in HCR to allow HCR amplification of multiple target biomolecules.
The method of claim 36, wherein the orthogonal HCR initiators are directly conjugated to the orthogonal antibodies using chemical linkers, said chemical linker is selected from an amine-reactive linker containing a succinimidyl ester group, a thiol-reactive linker or a click chemistry linker.
The method of claim 37, wherein the click chemistry linker is selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker.
The method of claim 37 or 38, wherein the antibody is a secondary antibody that reacts with a primary antibody specific to an analyte, the secondary antibody is a IgG or a Nanobody, and the primary antibody is a IgG, a Nanobody or a scFv.
The method of claim 36, wherein the orthogonal binders are genetically-engineered protein tags for labeling different target biomolecules, and the orthogonal HCR initiators are conjugated to tag binding partners respectively, which are capable of binding tags.
The method of claim 40, wherein the tag has a chemical group nonreactive toward a biomolecule, said chemical group is selected from an amine moiety, a carboxyl moietty, a thiol moiety and a glycosylated modification moiety.
The method of claim 40 or claim 41, wherein the HCR initiators are conjugated to tag binding partners selected from SpyCatcher, SnoopCatcher, benzylguanine (BG) , and scFv, and subsequently are used to detect the subcellular localization of the genetically-encoded tags selected from SpyTag, SnoopTag, SNAP-tag, and GCN4-tag respectively.
The method of claim 40 or claim 41, wherein the tag is CLIP-tag or Halo-tag.