Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Mammalian Cells

doi:10.21203/rs.3.pex-895/v1

Method Article

Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Mammalian Cells

https://doi.org/10.21203/rs.3.pex-895/v1

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We developed a novel auto-nucleation suppressed mechanism (ANSM) for direct synthesis of EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags (e.g., metallothionein) in cells for single-molecule detection with electron microscopy (it accompanies our Nature Method manuscript, Jiang et al. 2020 ^[1]). Both tagged-fusion proteins expressed in cells (e.g.bacteria, yeast and mammalian cells) and antigens stained with antibody-tag fusion proteins can be visualized by this protocol. Here we describe the typical protocols (both the chemical fixation and the high pressure freezing cases) developed for ANSM-based AuNP synthesis in HeLa cells expressing metallothionein (MTn) tags (Figure 1). This approach should be widely applicable to many systems for EM visualization of single-molecule in mammalian cells.

Biological techniques

Biotechnology

Cell Biology

Biophysics

Cysteine-rich tag

electron microscopy

gold nanoparticle

mammalian cell

metallothionein

AuNP synthesis

single-molecule

HeLa cell

oxidization

chemical fixation

high pressure freezing

freeze-substitution fixation

gold enhancement

antibody

imaging

ultrastructure

labeling

G418 (GIBCO),hygromycin B(GIBCO),Trypsin-EDTA (GIBCO); Dulbecco’s modified Eagle medium (DMEM)(GIBCO); Fetal bovine serum (FBS) (GIBCO);2-mercaptoethanol (2-ME, Cat.0482-250ML, Amresco); HAuCl₄(Cat.4022-1G, Sigma); NaBH₄(Sigma); D-penicillamine (D-P) (Cat.P0147, TCI); Tris(2-carboxyethyl)phosphine hydrochloride (TCEP×HCl) (Cat.646547, Sigma); 3,3'-dithiodipropionic acid (DTDPA)(Cat.D0947, TCI);S-methyl methanethioslfonate (MMTS)(Cat.401398, J&K scientific);HEPES(Sigma); PIPES(Sigma); 25% glutaraldehyde solution (Cat.16220, Electron Microscopy Sciences); 10% Glutaraldehyde in acetone (Cat.16530, Electron Microscopy Sciences);OsO₄(Cat.19110, EMS) ; Uranyl acetate (Cat.22400, EMS); sorbitol (Sigma); glycine(Sigma); Triton X-100 (Sigma); Zymolyase-20T (Cat.320921, MP); Methanol; Acetone (>99.7%).

35 mm cell culture dishes (Corning); 6-well cell culture plates(Corning);50mL polypropylene centrifuge tubes; Customized 3.05 mm x 0.66 mm specimen holders for HPF (Beijing Wulundes Biotech Ltd.); 3mmx0.16mm sapphire discs (Beijing Wulundes Biotech Ltd., or Engineering Office of M. Wohlwend GmbH); 3mm 0.025mm/0.275mm Aluminum carrier (Beijing Wulundes Biotech Ltd., Engineering Office of M. Wohlwend GmbH);2mL polypropylene screw cap microtubes (Cat. 81-0204, Biologix);1.5mL MaxyClear snaplock microtube (MCT150C, Axygen Scientific); Flat mold or flat-tip BEEM embedding capsule (Tedpella Inc.); 200 mesh hexagonal copper grid (G200HEX, Tedpella Inc.);solvent resistant pen (Cat.No.62053-B, EMS); SPI-Pon 812 resin (SPI Inc.);Formvar 15/95 resin (EMS);Foam cryobox; Forceps; Tweezers; Pasteur pipette; Liquid nitrogen (LN₂); 4L Dewar for LN₂;Alcohol lamp;2 pairs of 45-degree fine-tipped tweezers (Ted Pella, Inc.); Filter paper (Fisher Scientific, Whatman No. 1); Paper points (Ted Pella, Inc.).

Reagent Setup:

PBS-A buffer: Dissolve NaH₂PO₄(1.125 mM), Na₂HPO₄(3.867 mM), NaCl (100 mM) in 1L ddH₂O, adjust to pH 7.4.

10X PBS buffer: Dissolve NaH₂PO₄(100mM), K₂HPO₄(18 mM), KCl (27 mM), NaCl (1.37 M) in 1L ddH₂O.

1X PBS buffer: dilute 10X PBS to 1X PBS, and adjust pH to 7.4.

0.2 M HEPES buffer: Dissolve HEPES (0.2M) in 980mL of ddH₂O, then add 10 mL of 100mM MgCl₂and 10mL of 100mM CaCl₂(final concentration 1 mM) respectively, adjust pH to 7.4.

50mM glycine in 0.2 M HEPES buffer:Add 50mL of 1M glycine to 950 mL of 0.2M HEPES buffer (containing 1mM CaCl₂and 1 mM MgCl₂).

10% UA in methanol: 1g uranyl acetate dissolved in 10 mL of methanol in a 15mL centrifuge tube wrap with aluminum foil, vortex to dissolve, filtered and store at -20°C.

0.1M PIPES: 0.1 M PIPES buffer with 1 mM MgCl₂, 1 mM CaCl₂and 0.1 M sorbitol (pH 6.8).

Fresh 10 mM HAuCl₄in ddH₂O: make 10mM HAuCl₄in 1mL of ddH₂O in a 2mL polypropylenemicrotube, seal and wrap with aluminum foil, store at 4°C;

1% HAuCl₄in acetone: Dissolve 0.1g HAuCl₄in 1 mL of acetone in a 2mL microtube, seal and wrap with aluminum foil, store at -20°C.

Fresh 10 mM NaBH₄in acetone: (1) Weight 0.04g NaBH₄and sealed in a 2mL tube keep at -60°C; (2) Add 1 mL of pre-cooled to -60 °C methanol to tube to make 1 MNaBH₄in methanol; (3) Transfer 10 µL of 1 M NaBH₄to 1 mL of cold acetone at -60 °C to make 10 mMNaBH₄in acetone. Freshly prepared for immediate use only.

Fresh 100 mM NaBH₄in ddH₂O: (1) Weight 0.04g NaBH₄and sealed in a 2mL tube on ice; (2) Add 1 mL of ddH₂O to tube to make 1 MNaBH₄in ddH₂O; (3) Transfer 100 µL of 1 M NaBH₄to 900 µL of ddH₂O on ice to make 100 mMNaBH₄in ddH₂O. Freshly prepared for immediate use only.

1% OsO₄+ 0.1 % UA in acetone: (1) Add 24.5 mL of acetone into a 50mL polypropylene centrifuge tube and keep the tube closed on ice; (2) Open an ampule with 0.25 g OsO₄(wrap with several layers of paper towels); (3) Fetch 0.3 mL of acetone with a glass Pasteur pipette, add to the broken ampule and gently suck in and blow out to dissolve OsO₄, and then transfer the solution back to the 50mL tube. (3) Add 0.25 mL 10% of UA to the 50mL tube, and mix well; (4) Dispense the 1 % OsO₄+0.1 % UA in acetone into 2mL polypropylene crew cap microtubes by a glass Pasteur pipette (about 1 mL per tube, the 1 mL position of tube can be pre-labelled); (5) Cap the tubes immediately and then immerse into LN₂for freezing by holding the tube right-side up; (g) Store the frozen 1 % OsO₄+0.1 % UA fixative under LN₂until ready to use.

Laminar flow cabinet; Biosafety level 2 cabinet;

High-pressure freezing machines (Wohlwend HPF Compact 01, Leica HPM 100);

Leica UC7/FC7 ultramicrotome (Leica Microsystem);

Leica AFS2 freeze-substitution machine;

FEI Spirit electron microscope (FEI Corp.);

4k x 4k 895 CCD camera (Gatan Corp.);

Plasma cleaner (Harrick Plasma);

Vacuum oven (20-200 °C);

Dissecting microscope with fiber-optic light source.

1. Mammalian cell lines and cell culture (Timing 3 day)

The detailed information for preparing the stably transfected HeLa cell lines that expressing metallothionein (MTn) tags was described in our Nature Methodmanuscript (Jiang et al. 2020 ^[1]).The selected stable expression cells were cultured under selective growth conditions (e.g.DMEM with 10% FBS and200 mg/mL G418 (for Ost4 or KDEL transfected cells) or 50mg/mL hygromycin B (for mito-AcGFP transfected cells)at 37 °C 5% CO₂incubator).Here list the procedures for preparing HeLa cells cultured on sapphire discs for EM experiments:

1.1 Ultrasonic cleaning of 3mmx0.16mm sapphire discs in a 2mLmicrotubes with 1 mL ethanol for 5 min;

1.2 Burning each sapphire disc in the flame of an alcohol lamp inside a laminar flow cabinet for 5 s;

1.3 Marking one side of each sapphire disc with a number with a solvent resistant pen;

1.4 Placing sapphire discs on the bottom of a 35mm culture dish or a well of 6-well cell culture plate (with the mark side on the bottom surface) (Figure 2);

1.5 Sterilizing the cell culture dish or platewith sapphire discs (each side) under UV light for 30 min.

1.6 Aliquot cells into the cell culture dish or platewith sapphire discs and add proper amount DMEM with 10% FBS and200 mg/mL G418 or 50 mg/mL hygromycin B;

1.7 Placing the cell culture dish or plate in a cell culture incubator for cell growing;

1.8 When the cell confluency reaches full confluence, ready for further EM experiments.

2. Oxidization and Fixation (Timing 1-3 day)

Cells cultured on the sapphire discs will be fixed either with chemical fixation (Scheme 2) or cryo-fixation (e.g., high pressure freezing (HPF)) (Scheme 3) before using for AuNP synthesis (Figure 1). The procedures for two Schemes are described as following:

Scheme 2 (b,d) : oxidization and chemical fixation (Timing ~ 14 h):

2.1 Oxidization (at 4 °Cor at room temperature)

Scheme 2b: Change the medium in the 35mm cell culture dish with1mLof5mM (or 3mM)DTDPAinselectedbuffer (e.g.,PBS, 0.2M HEPES, 0.1M PIPES, or DMEM with 10% FBS, pH 7.4) for 30 min oxidization at 4°C (Note: HEPES and PIPES containing 1 mM CaCl₂and 1 mM MgCl₂).

Scheme 2d: Change the medium in the culture dish with1mLof25mMMMTSinPBS (pH 7.4) for 5 min oxidizing at room temperature.

2.2 Fixation

Add 4 µL of 25% GA (0.1% final conc.) to the culture dish for 5-30 min fixation at 4°C (Note: keep the oxidant and the medium unchanged; Simply suck away thesolution in the dish immediately for the next neutralization step.

2.3 Neutralization

Change the solution in the culture dish with 1 mL of 50mM glycine in PBS (or the selected buffer) for neutralization at 4 °Covernight; Simply suck away the solution in the tube for next step.

2.4 Permeabilization

Permeabilize with 1 mL of 0.1% triton X-100 in PBS (or the selected buffer) in the culture dish at room temperature (RT) for 2 min;

2.5 Wash with 1 mL of PBS-A for 3 X 5 min; Ready for ANSM-based AuNP synthesis in the following step 3.1.

Scheme 3 (a,b,c) : high pressure freezing and freeze-substitution fixation (HPF/FSF) (Timing 40-53 h):

2.1 high pressure freezing (Timing ~ 1 h)

(1) Fill the pre-cleaned 0.025 mm deep aluminum carrier with 2 µL of 1-hexadecene;

(2) Pick a 3 mm x 0.16 mm sapphire disc (from the above step 1.8) with forceps and quickly cap the disc on to the 0.025 mm deep aluminum carrier with the cells facing the cavity (distinguish by the label);

(3) Load the capped carrier into the customized 3.05 mm x 0.66 mm HPF specimen holder for high pressure freezing;

(4) Unload the carrier under liquid nitrogen in the foam cryobox and stored in 2mL polypropylene microcentrifuge tubes in liquid nitrogen before use.

2.2 Freeze-substitution fixation (Timing 39-52 h)

Load 1-4 specimens (attached on sapphire discs) into a 2mL polypropylene microtube (we also tried 35mm polypropylene dish, see the Trouble Shooting section) with 1 mL of frozen freeze-substitution solvent (different for following Scheme 3a,3b,3c) under liquid nitrogen(LN₂) with a pair of pre-cooled forceps; Then transfer the microtube to the pre-cooled to -90°C chamber of a Leica AFS2 freeze-substitution machine for FSF processing.

The procedures for Scheme 3a,3b,3c are described as following:

Scheme 3a (HPF/FSF) (Timing 42 h):

(1) Keep the specimens in 1 mL of acetone containing 10 mM DTDPA + 5% ddH₂O + 1% methanol at -90°C for 12h;

(2) Warm up gradually from 90°C to -20°C within 8h;

(3) Stay at -20°C for 2h;

(4) Cool back from -20°C to -30°C within 1h;

(5) Add 10 µL of 10% GA (in acetone) to the microtube (still contains the 1mL of DTDPA solution) at -30°Cfor 5 h fixation;

(6) Gradual rehydration:

a. Change to pre-cooled -30 °C 30% ddH₂O in acetone, gradually warm up to -20 °C within 30 min;

b. Change to pre-cooled -20 °C 50% PBS in acetone, gradually warm up to 4 °C within 1h;

c. Change to pre-cooled 4 °C 70% PBS in acetone for 10 min;

d. Change to pre-cooled 4 °C PBS for 10 min;

(7) Neutralization: change to 1 mL of 50 mM glycine in PBS for neutralization at 4 °Covernight;

(8) Wash with 1 mL PBS-A for 3 X 5 min; Ready for ANSM-based AuNP synthesis in the following step 3.1.

Scheme 3b (HPF/FSF; Gold enhancing) (Timing 52 h):

(1) Keep the specimens in 1 mL of acetone at -90 °C for 3h;

(2) Change to 1 mL of acetone containing 50 mM DTDPA + 0.02% TA + 5% ddH₂O at -90°C for 12h;

(3) Warm up gradually from -90°C to -60°C within 5h;

(4) Stay at -60°C for 10h;

(5) Warm up gradually from -60°C to -35°C within 5h;

(6) Change to 0.1% GA + 0.01% UA + 50mM DTDPA+5% ddH₂O in acetonefor 5h fixation at -35°C;

(7) Change to 1 mL of acetone containing 30% 0.2M HEPES buffer for 30 min rehydration at -35°C;

(8) Warm up from -35°C to 0°C within 10 min;

(9) Replaced with 1mL of RT 0.2M HEPES buffer containing 50 mM glycine at 0°C for 5 min;

(10) Neutralize excess aldehyde with 1 mL of 50 mM glycine in PBS at 4 °C overnight;

(11) Wash with 1 mL of PBS-A for 3 X 5 min ; Ready for ANSM-based AuNP synthesis in the following step 3.1.

Scheme 3c (HPF/FSF; No oxidizing + No GA fixation) (Timing ~ 39 h):

(1) Keep the specimens in 1 mL of acetone for 2h at -90 °C;

(2) Change to 1 mL of acetone containing 0.02% TA at -90°C for 8h (or 0.01% TA for 12 h);

(3) Warm up gradually from -90°C to -60°C within 3h;

(4) Wash with acetone for 3x1h at -60°C;

(5) Warm up gradually from -60°C to -30°C within 3h;

(6) Add 2µLof 10% UA (final concentration: 0.01% UA) in 1 mL of acetonefor 3h fixation at -30°C;

(7) Wash with acetone for 3x30min at -30°C;

(8) Warm up gradually from -30°C to 4°C within 2h;

(9) Instant or gradual rehydration

Take out the sapphire disc (with cells attached on), immediately drop into a 35mm culture dish containing 1 mL of 0.2M HEPES with 1mM CaCl₂and 1 mM MgCl₂(pH 7.4) for instant rehydration; Or drop into 70% acetone in 0.2M HEPES with 1mM CaCl₂and 1 mM MgCl₂(pH 7.4) for a transitionalrehydration.

(10)Change to 0.2 M HEPES with 1 mM CaCl₂and 1 mM MgCl₂(pH 7.4)at 4 °C overnight;

(11) Wash with 1 mL of PBS-A for 3 X 5 min; Ready for ANSM-based AuNP synthesis in the following step 3.1.

3. ANSM-based AuNP synthesis for mammalian cells (Timing 3 h)

For cell grown on sapphire disc case:

3.1 Transfer 1-4 sapphire discs to a 35mm culture dish containing 1 mL of PBS-A buffer at RT (keep the marked side of the disc on the bottom).

3.2 Add 4.28 µL of 2-ME into the culture dish at RT incubate for 1 h;

3.3 Add 4.28 µL of 2-ME into a 2mL polypropylene microtube containing 1 mL of PBS-A buffer (pH 7.4), add 80 µL of 10 mM HAuCl₄, immediately vortex; Add 80 µL of 500 mM D-P, vortex; Change the solution in culture dish with this mixture and incubate for 2hat 4 °C;

3.4 Add 20 µL of 100 mM NaBH₄, immediately shake to mix well, incubate for 5 min (Note: 100 mM NaBH₄must be freshly prepared at 4 °C for immediate use).

3.5 Change solution to 1 mL of PBS-A at 4 °C.

For cell in suspension case (Note: only tried with Scheme 2b(PBS):

3.1 Disperse the cell pellet with 1 mL of RT PBS-A buffer in 2mL polypropylene microtube.

3.2 Add 4.28 µL of 2-ME into the microtube at RT, and then incubate for 1 h;

3.3 Add 80 µL of 10 mM HAuCl₄to the microtube, immediately vortex; Add 80 µL of 500 mM D-P, vortex; Then, incubate for 2hat 4 °C;

3.4 Add 20 µL of 100 mM NaBH₄, immediately vortex to mix well, incubate for 5 min (Note: 100 mM NaBH₄must be freshly prepared at 4 °C for immediate use).

3.5 Centrifuge at 800 rpm for 2 min to obtain cell pellet;

3.6 Change solution to 1 mL of PBS-A at 4 °C.

(Note: for suspension case, the rest standard HPF/FSF and embedding all need similar adjustments: centrifugation 800 rpm X 2 min to obtain cell pellet, and load into aluminum carrier for freezing).

4. HPF/FSF (Timing 56-58 h):

4.1 high pressure freezing (Timing 1 h)

(1) Fill the pre-cleaned 0.025 mm deep aluminum carrier with 2 µL of 1-hexadecene;

(2) Pick a 3 mm x 0.16 mm sapphire disc (from the above step 3.4) with forceps and quickly cap the disc on to a 0.025 mm deep aluminum carrier with the cells facing the cavity (distinguish by the label);

(3) Load the capped carrier into the customized 3.05 x 0.66 mm HPF specimen holder for high pressure freezing;

(4) Unload the carrier under liquid nitrogen in the foam cryobox and stored in 2mL polypropylene microcentrifuge tubes in liquid nitrogen before use.

4.2 Freeze-substitution fixation and gold enhancing (Timing 56-58 h):

After the AuNP synthesis, the frozen specimens were either processed with standard 1% OsO4+0.1% UA in acetone freeze-substitution fixation directly or with optional FSF-based gold enhancement (Scheme 3b; Figure 2) prior to the standard 1% OsO4+0.1% UA in acetone freeze-substitution fixation.

Standard FSF with 1% OsO4+0.1% UA in acetone (Timing 56 h):

(1) Load 1-4 specimens (attached on sapphire discs) into a 2mL polypropylene microtube with 1 mL of frozen acetone containing 1% OsO₄+0.1% UA under LN₂;

(2) Transfer the microtube to the pre-cooled to -90°C chamber of a Leica AFS2 freeze-substitution machine and stay for 24h at -90 °C;

(3) Warm up gradually from -90°C to -60°C within 5h;

(4) Hold at -60°C for 6h;

(5) Warm up gradually from -60°C to -20°C within 3h;

(6) Hold at -20°C for 18h;

(7) Warm up gradually from -20°C to 4°C within 2h;

(8) Wash with acetone 3 x 1 h at 4 °C;

(9) Warm up to RT for resin infiltration;

Scheme 3b (FSF-based gold enhancing) (Timing 58 h):

(1) Load 1-4 specimens (attached on sapphire discs) into a 2mL polypropylene microtube with 1 mL of frozen acetone containing 0.5% GA under LN₂;

(2) Transfer the microtube to the pre-cooled to -90°C chamber of a Leica AFS2 freeze-substitution machine and stay for 3h at -90°C;

(3) Warm up gradually from -90°C to -60°C within 6h;

(4) Wash with acetone for 3x1h at -60°C;

(5) FSF-based gold enhancing

(a) Add 10µL of 1% HAuCl₄(in acetone) to the microtube containing 1mL of acetone for 8h incubation at -60°C;

(b) Add 10 µL of fresh 10 mM NaBH₄(must be fresh prepared) to the solution for 20h gold enhancing at -60°C;

(6) Change to 1 mL of 1%OsO4+0.1% UA in acetone for 5h fixation at -60°C;

(7) Warm up gradually from -60°C to -30°C within 5h;

(8) Warm up gradually from -30°C to 4°C within 2h;

(9) Wash with acetone 3x1h at 4°C;

(10)Warm up to RT for resin infiltration;

5. Resin infiltration, embedding, polymerization, and thin sectioning (Timing ~3 day):

5.1 Infiltrate the sample with 1:1 SPI-Pon 812 resin: acetone for 1.5h, 3:1 SPI-Pon 812 resin:acetone for 6-12h, 100% SPI-Pon 812 resin for 1 h, 100% SPI-Pon 812 resin for 3 h, and 100% SPI-Pon 812 resin for 1 h on a rotator.

5.2 Transfer the resin infiltrated sample attached on the sapphire disc to a flat-tip BEEM embedding capsule, incubate in fresh resin for 12 h polymerization at 45°C followed with 24-48h at 60°C(Note: the side with cells should be kept right-side up, see Figure 2).

5.3 Trim the polymerized specimen carefully to exposure the sapphire disc, dip into LN₂for 10 s to detach the cells from the disc and separate the disc from the block with forceps; then trim the block to trapezoidal shape for thin sectioning (e.g., 90nm) with a Lecia ultramicrtome.

5.4 Pick the sections on a 200-mesh hexagonal copper grid that coated with/without formvar.

6. EM imaging of cells (Timing 6-24 h):

90 nm thin sections on copper grids positive stained with 2% UA for 6 min or without positive staining were directly used for EM imaging on an FEI Spirit electron microscope (FEI Corp.) operating at 120kV. The images were recorded with a 4k x 4k 895 CCD camera (Gatan Corp.) under proper defocus values. Usually at magnification range from 11kX to 30kX are suitable for visualizing AuNPs in cells with proper defocus values to ensure 2-3 nm AuNPs visible.

(1) For all processes, should take care of the sapphire discs for avoiding strong shaking to scratch the cells on the discs; We also tried to burn some round grooves with a hot 5mm metal cylinder on the bottom of a 35mm polypropylene dish for fitting sapphire discs for FSF to avoid scratches (using 2mL solution instead) and achieve better results.

(2) NaBH₄should be freshly made before use.

(3) For chemical fixation cells with GA, the fixation strength (concentration and fixation duration) sensitively affects the AuNP labeling density.

(4) For ANSM-based AuNP synthesis, when add HAuCl₄requires immediately vortex to mix with 2-ME rapidly (because 2-ME reacts with Au³⁺almost instantly, the local concentration does affect the ratio of thiolate/Au³⁺, which might form polymeric forms of Au(I)SR compounds).

(5) The current ANSM-based AuNP synthesis protocol was optimized for 1 mL solution, changing to other volume might need to re-optimize due to the reason mentioned in the above item (2).

(6) When no HPF/FSF equipment is available, the final HPF/FSF processes could be replaced by regular chemical fixation and dehydration or using freezer for FSF.

(7) The buffers used in this protocol were tested extensively, if change to other buffers or with additional chemicals, user should test carefully (e.g., we found additional sugar might affect final AuNP synthesis).

(8) We tested this protocol also working for tissue samples (e.g., C. elegans), however, tissue sample might need further optimization.

(9) For suspension samples (e.g., bacteria, yeast, isolated animal cells), the protocol requires some modifications, e.g., requires centrifugation to collect cells for many steps, change fixative strength and duration (See another separate protocol for yeast cells and also our Nature Method manuscript, Jiang et al. 2020 ^[1]for more details).

(10)For using tag-antibody immune-staining of antigens in cells (e.g., GBP-MTn stains GFP), see the following supplementary protocol (Also see our Nature Method manuscript, Jiang et al. 2020 ^[1]for more details).

SUPPLEMENTARY PROTOCOL FOR IMMUNOSTAINING GFP WITH GBP-MTN

1. Cell culture as described in Section 1.

2. Fixation

Remove cell culture medium and wash cells with 0.2 M HEPES once. Add 2 mL of 0.1% GA or 4% PFA in 0.2 M HEPES to the 35mm culture dish for 5 min fixation at RT;

3. Neutralization

Change the solution in the culture dish with 2 mL of 50 mM glycine in 0.2 M HEPES for neutralization at 4 °Covernight; Simply suck away the solution in the dish for next step.

4. Permeabilization

Permeabilize with 2 mL of 0.05% triton X-100 in 0.2 M HEPES in the culture dish at room temperaturefor 2 min.

5. Blocking

Block cells in antibody blocking solution (0.25% cold fish gelatin and 0.1% BSA in 0.2 M HEPES) for 2h at RT.

6. Immunolabling

Incubate cells with 2 mL of 50 µg/mL GBP-MTn in blocking solution at 4 °C overnight;

Wash with 2 mL of 0.2 M HEPES for 5 min x 3;

Wash with 2 mL of PBS-A for 5 min x 3;

Ready for the rest standard steps are the same as the above Section 3-6.

The whole processes from cell culture to final EM imaging of the above protocol require 11-13 days.

Two example images from Scheme 3c were shown inFigure 3.

[1] Jiang,Z., Jin X., Li,Y., Liu, S.,Wang, Y.-Y., Zhao,P.,Cai,X., Liu Y., Tang,Y., Sun,X., Liu,Y., Hu,Y., Li,M., Cai,G., Qi,X., Chen,S., Du,L.-L. and He,W.,Direct synthesis of EM-visible gold nanoparticles on genetically encoded tags for single-molecule visualization in cells, Nature Methods manuscript(2020).

The National Institute of Biological Sciences, Beijing, filed a PCT patent (WO2019024707A1) covering part of the information contained in this article.

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Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Mammalian Cells

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Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Mammalian Cells

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