WO2019144391A1 - Utilisation d'ishcr pour l'élimination de tissu à base d'exm et de solvant - Google Patents

Utilisation d'ishcr pour l'élimination de tissu à base d'exm et de solvant Download PDF

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WO2019144391A1
WO2019144391A1 PCT/CN2018/074365 CN2018074365W WO2019144391A1 WO 2019144391 A1 WO2019144391 A1 WO 2019144391A1 CN 2018074365 W CN2018074365 W CN 2018074365W WO 2019144391 A1 WO2019144391 A1 WO 2019144391A1
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hcr
linker
antibody
tag
orthogonal
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PCT/CN2018/074365
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Rui Lin
Minmin LUO
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National Institute Of Biological Sciences, Beijing
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens

Definitions

  • 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) ) .
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • Fluorogenic substrates especially those based on HRP-tyramide reaction chemistries, have been developed to support high-resolution fluorescence microscopy 3 .
  • 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.
  • HCR hybridization chain reaction
  • nucleic acid probes complementary to the target mRNA molecule are used as ‘initiator’ oligos.
  • 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.
  • ExM expansion microscopy
  • 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.
  • 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.
  • HCR Hybridization Chain Reaction
  • 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.
  • HCR Hybridization Chain Reaction
  • 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.
  • moieties such as acrydite, amine, acrylamide, 6- ( (acryloyl) amino) hexanoic acid or methacrylic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the binder can be an antibody, a fragment of an antibody, or a genetically-engineered protein tag.
  • the orthogonal binders are orthogonal antibodies
  • the antibodies may be biotinylated antibodies
  • 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.
  • 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.
  • 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.
  • 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
  • the primary antibody may be a IgG, aNanobody or a scFv.
  • 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.
  • 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.
  • 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) .
  • subcellular locations e.g., cell and vesicle membrane, cytosol, mitochondrion, and cell nucleus
  • 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.
  • 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.
  • 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.
  • 3DISCO-based tissue-clearing methods such as uDISCO and iDISCO
  • HCR Hybridization Chain Reaction
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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 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 present method is compatible with 3DISCO-based tissue-clearing methods, such as uDISCO and iDISCO.
  • 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.
  • 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.
  • a chemical group is selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazine, Alkyne, and/or DBCO
  • the said fluorescent dye
  • a pair of fluorophore-tagged amplifiers are added to the final round of the multiple-round HCR for visualization.
  • 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.
  • the binder can be an antibody, a fragment of an antibody, or a genetically-engineered protein tag.
  • the orthogonal binders are orthogonal antibodies
  • the antibodies may be biotinylated antibodies
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • a type of HCR initiator capable of both binding to the target and anchoring itself to the swellable polymer is designed.
  • 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) .
  • DNA-fluorophore HCR amplifiers were applied.
  • 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 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
  • the staining is performed after clearing.
  • our isHCR should also be compatible with these aqueous solutions-based clearing methods.
  • 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 H ybridization C hain R eaction.
  • 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.
  • HCR hybridization Chain Reaction
  • Click chemistry is a class o f biocompatib l e rea c tions 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 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
  • 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°C 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.
  • 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 2 O and stored at -20°C.
  • 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°C until use.
  • 4-hydroxy-TEMPO (4-HT, 176141)
  • Ammonium persulfate (APS, A3678)
  • Tetramethylethylenediamine (TEMED, T7024)
  • sodium acrylate 408220
  • DBE DiBenzyl Ether
  • D8906 Dextran sulfate
  • 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.
  • 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) .
  • 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.
  • DTT dithiothreitol
  • 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) .
  • 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) .
  • 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°C. Tissue samples were first dehydrated in 30%sucrose solution for preparing thin sections (50 ⁇ m) or, for large volume tissue samples (lungs and brain sections thicker than 500 ⁇ m) , pretreated with methanol according to the original iDISCO+ protocol.
  • PFA paraformaldehyde
  • mice brain sections were 50 ⁇ m for immunofluorescent labeling or for expansion microscopy (ExM) .
  • Thin sections were prepared on a Cryostat microtome (Leica CM1950) .
  • thick sections 500 ⁇ m 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.
  • HCR amplification buffer [5 ⁇ sodium chloride citrate (SCC buffer) , 0.1%vol/vol Tween-20, and 10%wt/vol dextran sulfate in ddH 2 O] .
  • SCC buffer sodium chloride citrate
  • PBST PBST
  • 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.
  • biotin 5 ng ⁇ mL -1 , 30 min at room temperature
  • biotin 5 ng ⁇ mL -1 , 30 min at room temperature
  • the second biotinylated secondary antibody was added and then amplified.
  • 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) .
  • SNAP-tag genetically encoded tags
  • SpyTag genetically encoded tags
  • GFP GFP
  • smFP_GCN4 HCR initiators
  • Fig. 3 After membrane permeabilization, cultured-cell or brain-section samples were incubated with appropriate binding partners.
  • SNAP-tag labeling we applied 0.1 ⁇ M BG-labeled HCR initiators or 0.5 -1 ⁇ M SNAP-Surface Alexa Fluor 546 and incubated these samples at room temperature for 1h.
  • SpyTag labeling we applied 25 ⁇ M HCR initiator-labeled SpyCatcher and incubated these samples at room temperature for 2h.
  • 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 2 O] .
  • SCC buffer sodium chloride citrate
  • a pair of DNA-fluorophore HCR amplifiers were snap-cooled separately in 5 ⁇ SSC buffer by heating at 95°C for 90s and cooling to room temperature over 30 min.
  • 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.
  • an additional graphene oxide step was added to this basic process for applications that demands background suppression.
  • GO (20 ⁇ g ⁇ 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.
  • 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 ⁇ g ⁇ 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.
  • ExM Expansion microscopy
  • the ExM protocols were performed following the original publications 7, 40 .
  • 50 ⁇ m 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°C. Sections were then transferred to a gelling chamber and gelled at 37°C for 2 h.
  • 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 2 O) at 35°C. Finally, samples were incubated in DiBenzyl Ether (DBE) at room temperature until clear.
  • DBE DiBenzyl Ether
  • 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.
  • FWHM full width at half maximum
  • ⁇ is the standard deviation of the fitted Gaussian curve.
  • the mean intensity of the distance from the edge of the membrane or neuronal process (typically between -3 ⁇ m to -2 ⁇ m) 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.
  • Vglut3-immunopositive puncta 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.
  • 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.
  • biotin and acrydite dual-labeled HCR initiators are used. HCR amplifiers are applied after gelation and digestion.
  • HCR amplifier incubation 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.
  • 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.

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Abstract

L'invention concerne un procédé d'optimisation d'isHCR pour ExM, et un isHCR optimisé pour un procédé d'élimination de tissu à base de 3 DISCO.
PCT/CN2018/074365 2018-01-26 2018-01-26 Utilisation d'ishcr pour l'élimination de tissu à base d'exm et de solvant WO2019144391A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021113505A1 (fr) * 2019-12-05 2021-06-10 Massachusetts Institute Of Technology Procédé de préparation d'un échantillon pour la microscopie à expansion
US11180804B2 (en) 2017-07-25 2021-11-23 Massachusetts Institute Of Technology In situ ATAC sequencing
US11408890B2 (en) 2015-04-14 2022-08-09 Massachusetts Institute Of Technology Iterative expansion microscopy
US11802872B2 (en) 2017-02-24 2023-10-31 Massachusetts Institute Of Technology Methods for examining podocyte foot processes in human renal samples using conventional optical microscopy
US11873374B2 (en) 2018-02-06 2024-01-16 Massachusetts Institute Of Technology Swellable and structurally homogenous hydrogels and methods of use thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060234261A1 (en) * 2005-03-08 2006-10-19 Pierce Niles A Colorimetric readout of hybridization chain reaction
CN102196773A (zh) * 2008-10-23 2011-09-21 皇家飞利浦电子股份有限公司 分子成像
CN106170564A (zh) * 2014-02-04 2016-11-30 欧凌科生物科技公司 基于杂交链式反应(hcr)的检测的邻近试验

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060234261A1 (en) * 2005-03-08 2006-10-19 Pierce Niles A Colorimetric readout of hybridization chain reaction
CN102196773A (zh) * 2008-10-23 2011-09-21 皇家飞利浦电子股份有限公司 分子成像
CN106170564A (zh) * 2014-02-04 2016-11-30 欧凌科生物科技公司 基于杂交链式反应(hcr)的检测的邻近试验

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11408890B2 (en) 2015-04-14 2022-08-09 Massachusetts Institute Of Technology Iterative expansion microscopy
US11802872B2 (en) 2017-02-24 2023-10-31 Massachusetts Institute Of Technology Methods for examining podocyte foot processes in human renal samples using conventional optical microscopy
US11180804B2 (en) 2017-07-25 2021-11-23 Massachusetts Institute Of Technology In situ ATAC sequencing
US11873374B2 (en) 2018-02-06 2024-01-16 Massachusetts Institute Of Technology Swellable and structurally homogenous hydrogels and methods of use thereof
WO2021113505A1 (fr) * 2019-12-05 2021-06-10 Massachusetts Institute Of Technology Procédé de préparation d'un échantillon pour la microscopie à expansion
US11802822B2 (en) 2019-12-05 2023-10-31 Massachusetts Institute Of Technology Multiplexed expansion (MultiExM) pathology

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