Staining of Membrane Receptors with Fluorescently-labeled DNA Aptamers for Super-resolution Imaging

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Feb 2017


One of the most prominent applications of fluorescent super-resolution microscopy is the study of nanodomain arrangements of receptors and the endocytic pathway. Staining methods are becoming crucial for answering questions on the nanoscale, therefore, the use of small and monovalent affinity probes is of great interest in super-resolution microscopy with biological samples. One kind of affinity probe is the aptamer. Aptamers are single DNA or RNA sequences that bind with high affinity to their targets and due to their small size they are able to (i) place the fluorophore in close proximity to the protein of interest and (ii) bind to most of the protein of interest overcoming the steric hindrance effect, resulting in better staining density. Here we describe a detailed protocol with which to stain live cells using aptamers and to image them with Stimulated Emission Depletion (STED) microscopy. In this protocol, the stainings were performed with commercially available aptamers that target the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2 or ErbB2) and the ephrin type-A receptor 2 (Epha2). Since aptamers can be coupled to most of the popular fluorophores, we believe that the procedure presented here can be extended to the large majority of the current super-resolution microscopy techniques.

Keywords: Microscopy (显微镜), Super-resolution (超分辨率), STED (STED), Aptamers (适配子), Affinity probes (亲和力探针)


Recent advances in super-resolution imaging techniques have led to the search for more accurate methodologies to tag cellular elements. Diffraction unlimited imaging instruments provide excellent resolutions, however standard sample staining methodologies, such as immunostaining, lack the necessary precision for the detection of cellular elements. Due to their large dimension (~15 nm in length) and high molecular weight (~150 kDa), antibodies can poorly penetrate into biological samples. Additionally, the primary/secondary antibody complex places the fluorophores at approximately 25 nm away from the target, compromising the detection accuracy. Moreover, due to the large size of the primary/secondary antibody complex, a smaller fraction of the targets can be labelled due to the steric hindrance (Fornasiero and Opazo, 2015). This leads to lower labelling density, a crucial parameter for super-resolution microscopy, especially in recognizing and describing nanostructures. To circumvent these problems, small affinity probes that bind to single targets (monovalently) such as aptamers, affibodies or nanobodies have been tested in recent years (Rothbauer et al., 2006; Opazo et al., 2012; Ries et al., 2012) and are becoming valuable tools for super-resolution microscopy. Nanobodies, affibodies and aptamers have a relatively small linear size (~3 nm, ~2 nm and ~3 nm, respectively). This property allows them to place the fluorescent dye in close proximity to the target, penetrate samples in a more efficient manner and bind to a higher fraction of target proteins, bypassing the effect of steric hindrance observed by antibodies (Ries et al., 2012; Mikhaylova et al., 2015). In a previous study we made a systematic comparison between three commercially available aptamers and antibodies that target the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2 or ErbB2) and the ephrin type-A receptor 2 (Epha2). Our results showed that aptamers were able to find more epitopes (resulting in higher labeling density). As a consequence, several structural features of the subcellular components that were imaged became more apparent. Among these, the inner lumen and the complex morphology of endocytic organelles were visible. For these reasons, smaller imaging tools are becoming the preferred choice over antibodies, in order to improve the quality of an immunolabeling super-resolution approach and allow a more precise description the localization and the distribution of membrane receptors (Gomes de Castro et al., 2017).

Materials and Reagents

  1. Biospin 6 column (Micro Bio-SpinTM P-6 Gel Columns, Tris Buffer) (Bio-Rad Laboratories, catalog number: 7326222 )
  2. Cell culture 12-well plates (Thermo Fisher Scientific, catalog number: 150628 )
  3. 18 mm diameter round glass coverslips (Gerhard Menzel, catalog number: CB00180RA1 )
  4. 15 ml tubes
  5. PCR tubes
  6. Parafilm M
  7. Soft paper tissue
  8. Gloves
  9. Microscopy glass slides
  10. 2 ml tubes
  11. Vacuum filtration (e.g., VWR® Vacuum Filtration Systems, Standard Line) (VWR, catalog number: 10040-436 )
  12. 0.2 µm syringe filter
  13. Transfer pipette
  14. The images shown in Figure 1 are from aptamers against human:
    EGFR (5’-SH-EGFR aptamer-3’, seq # 2369-27-02, 50 mer)
    ErbB2 (5’-SH-ErbB2 aptamer-3’, seq # 1194 ± 35, 40 mer)
    Epha2 (5’-SH-EphA2 aptamer-3’, seq # 2176-01-01, 76 mer)
    Note: They were produced by Aptamer Sciences, Inc., South Korea and supplied by AMS Biotechnology, Europe. All three aptamers contain the chemical modification 5-(N-benzylcarboxyamide)-2’-deoxyuridine (5-BzdU) in unrevealed locations.
  15. Triethylammonium acetate buffer pH 7.0, 1 M (TEAA) (AppliChem, catalog number: A3846 )
  16. Maleimide Atto647N dye (Atto-TEC, catalog number: AD 647N-41 )
  17. Dimethyl sulfoxide, anhydrous (DMSO) (Sigma-Aldrich, catalog number: 276855 )
  18. Sodium chloride (NaCl)
  19. Ethanol
  20. 1x Dulbecco’s phosphate buffered saline (1x DPBS) (Sigma-Aldrich, catalog number: D8662 )
  21. Acetonitrile
  22. Trypsin-EDTA solution (Lonza, catalog number: 17-161E )
  23. Ultrapure DNase- and RNase-free distilled water (Carl Roth, catalog number: T143.2 )
  24. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Sigma-Aldrich, catalog number: C4706 )
  25. Sodium hydroxide (NaOH)
  26. DMEM, high glucose medium, no glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044 )
  27. Fetal bovine serum (FBS) (Biochrom, catalog number: S 0615 )
  28. L-Glutamine 200 mM (Lonza, catalog number: BE17-605E )
  29. Penicillin/streptomycin 10,000 U/ml, each (Lonza, catalog number: 17-602E )
  30. RPMI 1640 medium, no glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 21870076 )
  31. Poly-L-lysine (PLL) (Sigma-Aldrich, catalog number: P5899-5MG )
  32. Sodium phosphate dibasic (Na2HPO4)
  33. Potassium phosphate monobasic (KH2PO4)
  34. Potassium chloride (KCl)
  35. Magnesium chloride hexahydrate (MgCl2·6H2O)
  36. Salmon sperm DNA, sheared, 10 mg/ml (Thermo Fisher Scientific, catalog number: AM9680 )
  37. Dextran sulfate sodium salt (Sigma-Aldrich, catalog number: 31404 )
  38. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  39. Glycine (Sigma-Aldrich, catalog number: G8898 )
  40. Mowiol® (Sigma-Aldrich, catalog number: 81381 )
  41. 1 M Tris (2-carboxyethyl) phosphine hydrochloride stock solution (TCEP) (see Recipes)
  42. Complete DMEM medium (see Recipes)
  43. Complete RPMI medium (see Recipes)
  44. Poly-L-lysine (PLL) stock solution (see Recipes)
  45. 5x phosphate buffer saline (5x PBS) (see Recipes)
  46. 25 mM MgCl2 solution (5x MgCl2) (see Recipes)
  47. Blocking solution (see Recipes)
  48. 4% paraformaldehyde (PFA) (see Recipes)
  49. Quenching solution (see Recipes)
  50. Mowiol® (see Recipes)
  51. (Optional) Buffer A (see Recipes)
  52. (Optional) Buffer B (see Recipes)


  1. Microcentrifuge (e.g., Eppendorf, model: 5415 or similar)
  2. Nucleosil 100-5 C18 column
  3. (Optional) Dionex DNAPac PA200 4 x 250 mm column
  4. Hemocytometer
  5. Cell culture hood
  6. Thermal cycler
  7. Aluminum metal plate (length x width x thickness [cm]: e.g., 20 x 12 x 2)
  8. Cell culture incubator
  9. Half-curved-forceps
  10. Oven
  11. STED microscope, Leica pulsed STED setup composed by a True Confocal System (TCS) STED SP5 (Leica Microsystems, model: Leica TCS SP5 ) fluorescence microscope equipped with a 100x 1.4 NA HCX PL APO oil objective (Leica Microsystems, Germany)
  12. Pulsed laser (PicoQuant, Germany)
  13. Sapphire tunable laser (Mai Tai Broadband, Spectra-Physics, USA)
  14. Glass beaker
  15. Magnetic stirrer
  16. Lab coat, eye protection


  1. ImageJ (http://imagej.nih.gov/ij/docs/index.html)
  2. MATLAB (MathWorks, Massachusetts, USA)


  1. Coupling of dye to aptamers
    During this step a thiol-maleimide cross-linking reaction is performed to conjugate the fluorophore to aptamers. Alternatively, aptamers with a 3’-amino group can be used for further conjugation with NHS esters (e.g., Atto647N NHS-ester). Some fluorescently labeled aptamers are also commercially available.
    1. Add 10 nmoles of the thiolated aptamer with 10 mM Tris (2-carboxyethyl) phosphine solution (TCEP; see Recipe 1) in 100 μl of 0.1 M TEAA. Heat the tube at 70 °C for 3 min and incubate the mixture at room temperature for 1 h.
    2. Desalt this reaction by size exclusion chromatography on a Biospin 6 column according to the manufacturer’s instructions.
    3. Add to the reduced aptamer (from step A2) 4 µl of 10 µg/µl maleimide-functionalized Atto647N (previously dissolved in DMSO) and mix the contents well by pipetting or vortexing.
    4. Incubate overnight at 4 °C.
    5. Recover the fluorophore-labelled aptamer by ethanol precipitation: add 100 µl of 300 mM NaCl and 450 µl cold ethanol, mix carefully by inverting the closed sample several times and freeze the sample in dry ice for 20 min (or at -20 °C for several hours), centrifuge at 15,000 x g (rcf) at 4 °C for 30 min, discard the supernatant and wash the pellet in 50 µl of 70% ethanol.
    6. Resuspend in 50 µl 1x DPBS and desalt on a Biospin 6 column into 1x DPBS.
    7. Measure the absorbance ratio at 260 nm and 650 nm.
    8. Confirm the labeling efficiency and absence of free dye by reversed phase HLPC (e.g., on a Nucleosil 100-5 C18 4 x 250 mm column using a gradient of 0-40% acetonitrile in TEAA buffer in 30 min with a flow rate of 1 ml/min at 30 °C) or by anion exchange HPLC (e.g., Dionex DNAPac PA200 4 x 250 mm column using a gradient of 0-75% buffer B (see Recipe 12) in buffer A (see Recipe 11) in 40 min with a flow rate of 1 ml/min at 60 °C.
    9. Store the labeled aptamer sample at -20 °C until use.

  2. Cell culture preparation prior to staining
    Cell lines containing the receptor of interest (and cell lines lacking it, used as negative controls) should be seeded into 12-well plates containing coated coverslips with poly-L-lysine (PLL) one day before the aptamer staining. Depending on the cell line, choose adequate cell numbers (use the hemocytometer to count the cells) to reach approximately 70-80% confluence after 12-16 h incubation. The entire procedure described below must be performed under sterile conditions under a cell culture hood.
    1. HeLa (Epha2a positive) cells are grown in plates with complete DMEM medium (see Recipe 2) and A-431 (EGFR positive) and SKBR3 (ErbB2R positive) cells are cultured in complete RPMI medium (see Recipe 3). All cell lines are maintained at 37 °C and 5% CO2.
    2. To split cells, first wash cells with sterile 1x DPBS, add 2-3 ml of trypsin-EDTA to cover the surface of the plate and incubate the plate for 1-5 min at 37 °C or until cells are completely detached. Add 10 ml of complete DMEM medium or RPMI medium to inactivate trypsin. Gently pipette up and down the medium containing the inactivated trypsin to detach all cells from the plate and transfer them into a sterile 15 ml tube.
    3. Centrifuge the cells at 1,000 rpm (~250 x g) for 4 min at RT.
    4. Aspirate the supernatant and resuspend the cell pellet in 10 ml of complete DMEM medium/RPMI medium. Dilute the resuspended cells to the desired concentration in fresh complete DMEM or RPMI medium and add 1 ml to every well of the 12-well plates containing PLL treated coverslips (for PLL stock solution preparation and dilution, see Recipe 4). Incubate the plates at 37 °C and 5% CO2 until staining.

  3. Preparation of functional aptamer: folding reaction
    During this step, aptamers are exposed to a high temperature at an appropriate magnesium concentration which allows them to attain a proper folding prior to the staining procedure. For better results, the folding reaction should be freshly performed each time before staining. Before starting the folding reaction, prepare a stock solution diluting aptamers in 1x DPBS to a final concentration of 30 µM and store it protected from light at 4 °C.
    1. Prepare 10 µl of 10 µM functional aptamer by mixing 3.3 µl of fluorescently labelled aptamer (30 µM aptamer stock solution), 2 µl of 5x PBS (see Recipe 5), 2 µl of 5x MgCl2 (see Recipe 6) and 2.7 µl of ultrapure DNase- and RNase-free distilled water. Please note that this reaction should be carried out in PCR tubes that fit the thermal cycler.
    2. Heat up the aptamer solution to 75 °C for 3 min and then cool down to 20 °C at a rate of 1 °C/min using a thermal cycler.

  4. Aptamer live cells staining
    1. Aspirate the medium from the cells prepared during Procedure B, rinse briefly once with complete DMEM or RPMI depending on the cell line and incubate for 10 min at 37 °C and 5% CO2 (in the cell culture incubator) with 500 µl/well of freshly prepared blocking solution containing sheared salmon sperm DNA and dextran sulfate (see Recipe 7).
    2. In the meantime, prepare the metal plate used for staining/incubation at 37 °C. Fix with tape a piece of Parafilm® M (large enough to fit all coverslips to be stained) on the surface (see Video 1) and pre-heat the metal plate containing the Parafilm® M in the cell culture incubator.

      Video 1. Aptamers live staining part I. This video shows the metal plate preparation for aptamer live staining. Please note that this video is for demonstration only and that this step should be performed inside a cell culture hood.

    3. After blocking (step D1), carefully remove the coverslips from the 12-well plate using half-curved-forceps, remove the excess blocking solution by gently tapping the edges of the coverslips with a soft paper tissue and place the coverslips upside down on 60 µl of staining solution (complete DMEM supplemented with 100 μg/ml sheared salmon sperm DNA and 250 nM folded aptamer) spotted onto the Parafilm® M fixed to the metal plate (made in step D2) (see Video 2). Incubate the cells for 60 min at 37 °C and 5% CO2.

      Video 2. Aptamers live staining part II. This video shows removal of coverslips containing cells from the 12-well plates using half-curved-forceps and incubation of cells (on coverslips) with staining solution. Please note that this video is for demonstration only and that this step should be performed inside the cell culture hood. To avoid contamination, gloves should be sprayed with 70% ethanol.

    4. After incubation, carefully take the coverslips off the metal plate using half-curved-forceps, remove the excess staining solution by gently tapping the edges of the coverslips with a soft paper tissue, and then submerge it several times in large volumes (e.g., 20-40 ml in a small beaker) of ice-cold 1x DPBS. Briefly remove the excess 1x DPBS from the coverslips by tapping them on a tissue paper and place each one (cell-side-up) into a well of a new 12-well plate filled with 1 ml/well ice-cold 4% PFA (see Recipe 8) for fixation (see Video 3).

      Video 3. Aptamers live staining part III. This video shows the washing step after staining procedure. Please note that video is in open air for demonstration only and that 4% PFA should only be handled in the fume hood.

    5. Fix the cells for 20 min on ice and subsequently at room temperature for another 25 min. During fixation, keep the plate protected from light to avoid fluorophore bleaching.
    6. Aspirate the PFA solution and add 1 ml of quenching solution (see Recipe 9) to each well. Incubate for 15 min at room temperature protected from light.
    7. Wash twice for 5 min with 1x DPBS (1 ml/well) and mount the coverslips with Mowiol® (see Recipe 10; e.g., 8-9 µl for an 18 mm coverslip) on microscopy glass slides.
    8. Dry the mounted coverslips in an oven at 37 °C for 20-30 min or overnight at RT and store at 4 °C protected from light until imaging.

Data analysis

Imaging: The images shown in this protocol (Figure 1) were acquired using a 100x 1.4 NA HCX PL APO oil objective. Excitation of Atto647N fluorophore was achieved with a 635 nm pulsed laser (PicoQuant, Germany), and the 750 nm depletion STED beam was obtained with a pulsed infrared titanium: sapphire tunable laser (Mai Tai Broadband, Spectra-Physics, USA). The pixel size was set to 20.2 nm, scanning speed to 1 kHz, line average to 96 times, pinhole to one Airy unit and signal was detected with an avalanche photodiode detector (APD).

Figure 1. Recognition of endosome-like structures in aptamer or antibody stained cells. A. STED images comparing the cells stained with aptamers and antibodies against the same receptors (Ab1 and Ab2, under saturating conditions as described in (Gomes de Castro et al., 2017). Arrowheads point to some examples of endosome-like structures. Staining using antibodies resulted in discontinuous labelling of the organelle contours (indicated by yellow doted circles in the STED image). B. The scheme represents our current hypothesis, indicating that large affinity probes like antibodies (upper panel) might not detect all available epitopes and that small molecules like aptamers (lower panel) decorate the target structures better.


  1. To determine the saturating concentration or optimal staining concentration (best signal-to-noise ratio) for the aptamer of interest fixing the incubation time of staining (e.g., 60 min) and testing different aptamers concentrations ranging from 10-500 nM (or more) in positive cell lines is recommended. After staining with different concentrations of the aptamers, fixation and mounting, cells can be imaged in any epifluorescence microscope and fluorescent intensities can be calculated using as an example ImageJ or MATLAB.
  2. After defining the saturating conditions for the staining procedure, the next step is to test the binding specificity. This is achieved by evaluating the staining in the cells expressing the receptor and the cells that do not express it. Importantly, the staining conditions, the aptamer concentrations and the incubation times must be the same for both positive and negative cells. Once stained and fixed, cells can be imaged with an epifluorescence or confocal microscope. Negative cell lines should show virtually no specific fluorescent signal. For a proper control of the unspecific staining background, it is useful to test the immunolabeling with a random aptamer conjugated to the same fluorophore as the aptamer binding the specific target, in parallel. This control should not have any fluorescence signal. An additional control is the evaluation of the binding specificity by fluorescence flow cytometry analysis using the same controls.
  3. Every receptor has its own internalization kinetics. In this work and protocol we intend to maximize the labeling by internalization and therefore choose longer incubation times.
  4. If the aptamers contain BzdU (5-(N-benzylcarboxyamide)-2’-deoxyuridine) or any other hydrophobic groups, we recommend the addition of dextran sulfate to the blocking solution. An optimization of the dextran sulfate concentration in the pre-blocking solution strongly reduces the background caused by nonspecific binding due to electrostatic interaction of aptamers to the PLL-treated coverslip.
  5. If unspecific binding of the aptamer persists, increase the concentration of sheared salmon sperm DNA and/or dextran sulphate in the staining solution. The polyanionic competitor/blocking agent dextran sulfate also substantially reduces the nonspecific binding caused by electrostatic attraction of polyanionic aptamers to positively charged sites in nuclei, such as histones.
  6. For identification and quantification of endosome-like structures, colocalization studies are strongly recommended (Gomes de Castro et al., 2017).


  1. 1 M Tris (2-carboxyethyl) phosphine hydrochloride stock solution (TCEP)
    For 100 ml:
    1. Add 11.47 g TCEP to 35 ml cold ultrapure water
    2. Bring the pH to 7.0 with 10 N NaOH and adjust the volume to 100 ml
    3. Aliquot into 2.0 ml tubes and store at -20 °C
  2. Complete DMEM medium
    1. DMEM supplemented with 10% FBS, 4 mM L-glutamine and 100 U/ml each of penicillin and streptomycin
    2. Sterilize by vacuum filtration
    3. Store at 4 °C
  3. Complete RPMI medium
    1. RPMI supplemented with 10% FBS, 4 mM L-glutamine and 100 U/ml each of penicillin and streptomycin
    2. Sterilize by vacuum filtration
    3. Store at 4 °C
  4. Poly-L-lysine (PLL) stock solution
    1. Prepare 2 mg/ml stock solution in ultrapure water and sterilize through a syringe filter of 0.2 µm
    2. Make aliquots and store at -20 °C
    3. Prepare the plates containing PLL-treated coverslips under a sterile hood
    4. Add 1 ml of 0.1 mg/ml PLL into every well containing a coverslip
    5. Incubate for 1 h at RT
    6. After incubation, wash twice with ultrapure water and leave the plates to air-dry inside the cell culture hood
    7. Store the plates at 4 °C until use
  5. 5x phosphate buffer saline (5x PBS)
    1. Dilute the 10x concentrated PBS stock solution in ultrapure water. Filter the solutions inside a cell culture hood with a 0.2 µm syringe filter or autoclave. Store at RT
    2. For 1 L 10x concentrated PBS stock solution:
      14.4 g sodium phosphate
      2.4 g potassium phosphate
      2 g KCl
      80 g NaCl
      Dissolved in DNase-and RNase-free water
      Adjust the pH to 7.4
      Sterilize by filter sterilization or autoclaving
      Store at RT
  6. 25 mM MgCl2 solution (5x MgCl2)
    1. Dilute the 1 M MgCl2 stock solution to 25 mM in DNase- and RNase-free distilled water. To avoid particles and contamination, filter the solutions inside a cell culture hood with a 0.2 µm syringe filter. Store at room temperature
    2. For 100 ml of 1 M MgCl2 stock solution:
      Dissolve 20.3 g MgCl2·6H2O in 70 ml DNase-and RNase-free water and adjust the volume to 100 ml
      Store at RT
  7. Blocking solution
    DMEM or RPMI complete medium supplemented with 100 μg/ml sheared salmon sperm DNA and 1 mM dextran sulfate
  8. 4% paraformaldehyde (PFA)
    For 1 L PFA 4% preparation:
    1. Add approximately 600 ml of 1x PBS to 40 g of PFA in a glass beaker and stir using a magnetic stirrer at ~50 °C
    2. Adjust the pH to between 7 and 8
    3. Adjust the volume to 1 L with 1x PBS, make aliquots and store at -20 °C
    Note: PFA is highly toxic: use gloves, lab coat, respiratory and eye protection while handling PFA.
  9. Quenching solution
    1. 0.1 M glycine in 1x DPBS
      Store at RT until use
    2. To prepare 100 ml of glycine 1 M stock solution:
      Dissolve 7.5 g glycine in ultrapure water
      Filter it through a 0.2 µm syringe filter and store at RT
  10. Mowiol®
    1. Mix 24 g glycerol, 9.6 g Mowiol® 4-88 reagent, 62.4 ml distilled water and 9.6 ml 1 M Tris buffer in a conical cylinder with a magnetic stirrer for 5-7 days
    2. Optionally heat the mixture at 40-50 °C to dissolve Mowiol®
    3. After precipitation divide the supernatant in aliquots (e.g., in 2.0 ml tubes) and store at 4 °C
  11. (Optional) Buffer A
    25 mM Tris-HCl, pH 8
    6 M urea
  12. (Optional) Buffer B
    0.5 M NaClO4 in 25 mM Tris-HCl pH 8, 6 M urea


This protocol described here in more detail has been published in (Gomes de Castro et al., 2017). This work was supported by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).


  1. Fornasiero, E. F. and Opazo, F. (2015). Super-resolution imaging for cell biologists: concepts, applications, current challenges and developments. Bioessays 37(4): 436-451.
  2. Gomes de Castro, M. A., Hobartner, C. and Opazo, F. (2017). Aptamers provide superior stainings of cellular receptors studied under super-resolution microscopy. PLoS One 12(2): e0173050.
  3. Mikhaylova, M., Cloin, B. M., Finan, K., van den Berg, R., Teeuw, J., Kijanka, M. M., Sokolowski, M., Katrukha, E. A., Maidorn, M., Opazo, F., Moutel, S., Vantard, M., Perez, F., van Bergen en Henegouwen, P. M., Hoogenraad, C. C., Ewers, H. and Kapitein, L. C. (2015). Resolving bundled microtubules using anti-tubulin nanobodies. Nat Commun 6: 7933.
  4. Opazo, F., Levy, M., Byrom, M., Schafer, C., Geisler, C., Groemer, T. W., Ellington, A. D. and Rizzoli, S. O. (2012). Aptamers as potential tools for super-resolution microscopy. Nat Methods 9(10): 938-939.
  5. Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. and Ewers, H. (2012). A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9(6): 582-584.
  6. Rothbauer, U., Zolghadr, K., Tillib, S., Nowak, D., Schermelleh, L., Gahl, A., Backmann, N., Conrath, K., Muyldermans, S., Cardoso, M. C. and Leonhardt, H. (2006). Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3(11): 887-889.


【背景】超分辨率成像技术的最新进展已经导致搜索更精确的方法来标记细胞元件。衍射无限成像仪器提供优异的分辨率,然而标准样品染色方法,如免疫染色,缺乏检测细胞元素所必需的精度。由于它们的大尺寸(长度约15nm)和高分子量(〜150kDa),抗体可以很差地渗透到生物样品中。另外,一次/二次抗体复合物将荧光团放置在远离靶的大约25nm处,从而损害检测精度。此外,由于初级/二级抗体复合物的大尺寸,由于空间位阻(Fornasiero和Opazo,2015),较小部分的靶标可以被标记。这导致较低的标记密度,这是超分辨率显微镜的关键参数,特别是在识别和描述纳米结构时。为了克服这些问题,近年来已经测试了与单个靶(单价)结合的小的亲和力探针,如适体,亲和体或纳米体系(Rothbauer et al。,2006; Opazo et al。,2012; Ries et al。,2012 ),并且正在成为超分辨率显微镜的有价值的工具。纳米抗体,亲和体和适体具有相对较小的线性大小(分别为〜3nm,〜2nm和〜3nm)。这种性质允许它们将荧光染料置于靶附近,以更有效的方式穿透样品并且结合更高级别的靶蛋白,绕过由抗体观察到的空间位阻的作用(Ries等人,2012; Mikhaylova et al。,2015)。在以前的研究中,我们对靶向表皮生长因子受体(EGFR),人表皮生长因子受体2(HER2或ErbB2)和ephrin-A受体2(Epha2)的三种市售适配体和抗体进行了系统比较。 。我们的研究结果表明,适体能够找到更多的表位(导致更高的标记密度)。因此,成像的亚细胞组分的几个结构特征变得更加明显。其中,内腔和内细胞器的复杂形态是可见的。由于这些原因,较小的成像工具正在成为抗体的首选,为了提高免疫标记超分辨率方法的质量,并能更准确地描述膜受体的定位和分布(Gomes de Castro等, 2017年)。

关键字:显微镜, 超分辨率, STED, 适配子, 亲和力探针


  1. Biospin 6柱(Micro Bio-Spin< sup> P-6凝胶柱,Tris缓冲液)(Bio-Rad Laboratories,目录号:7326222)
  2. 细胞培养12孔板(Thermo Fisher Scientific,目录号:150628)
  3. 18毫米直径的圆形玻璃盖玻片(Gerhard Menzel,目录号:CB00180RA1)
  4. 15 ml管
  5. PCR管
  6. Parafilm M
  7. 软纸组织
  8. 手套
  9. 显微镜玻璃片
  10. 2 ml管
  11. 真空过滤(例如,真空过滤系统,标准生产线)(VWR,目录号:10040-436)
  12. 0.2μm注射器过滤器
  13. 转移移液管
  14. 图1所示的图像来自与人类的适配体:
    EGFR(5'-SH-EGFR aptamer-3',seq#2369-27-02,50mer)
    ErbB2(5'-SH-ErbB2 aptamer-3',seq#1194±35,40mer)
    Epha2(5'-SH-EphA2 aptamer-3',seq#2176-01-01,76 mer)
    注意:它们由韩国的Aptamer Sciences,Inc.生产,由欧洲的AMS生物技术公司提供。所有三个适体在不出现的位置含有化学修饰5-(N-苄基羧酰胺)-2'-脱氧尿苷(5-BzdU)。
  15. 三乙基乙酸铵缓冲液pH 7.0,1M(TEAA)(AppliChem,目录号:A3846)
  16. 马来酰亚胺Atto647N染料(Atto-TEC,目录号:AD 647N-41)
  17. 无水二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:276855)
  18. 氯化钠(NaCl)
  19. 乙醇
  20. 1x Dulbecco的磷酸盐缓冲盐水(1x DPBS)(Sigma-Aldrich,目录号:D8662)
  21. 乙腈
  22. 胰蛋白酶-EDTA溶液(Lonza,目录号:17-161E)
  23. 超纯DNA酶和无RNA酶的蒸馏水(Carl Roth,目录号:T143.2)
  24. 三(2-羧乙基)膦盐酸盐(TCEP)(Sigma-Aldrich,目录号:C4706)
  25. 氢氧化钠(NaOH)
  26. DMEM,高葡萄糖培养基,无谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:11960044)
  27. 胎牛血清(FBS)(Biochrom,目录号:S 0615)
  28. L-谷氨酰胺200mM(Lonza,目录号:BE17-605E)
  29. 青霉素/链霉素10,000 U / ml,各(Lonza,目录号:17-602E)
  30. RPMI 1640培养基,无谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:21870076)
  31. 聚-L-赖氨酸(PLL)(Sigma-Aldrich,目录号:P5899-5MG)
  32. 磷酸氢二钠(Na 2 HPO 4)
  33. 磷酸二氢钾(KH 2 PO 4)
  34. 氯化钾(KCl)
  35. 氯化镁六水合物(MgCl 2·6H 2 O)
  36. 鲑精子DNA,剪切,10mg / ml(Thermo Fisher Scientific,目录号:AM9680)
  37. 硫酸葡聚糖钠盐(Sigma-Aldrich,目录号:31404)
  38. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  39. 甘氨酸(Sigma-Aldrich,目录号:G8898)
  40. Mowiol ®(Sigma-Aldrich,目录号:81381)
  41. 1M Tris(2-羧乙基)膦盐酸盐储备溶液(TCEP)(参见食谱)
  42. 完成DMEM培养基(见食谱)
  43. 完成RPMI培养基(见食谱)
  44. 聚-L-赖氨酸(PLL)储备溶液(参见食谱)
  45. 5x磷酸缓冲盐水(5x PBS)(参见食谱)
  46. 25mM MgCl 2溶液(5x MgCl 2)(参见食谱)
  47. 阻塞解决方案(见配方)
  48. 4%多聚甲醛(PFA)(见配方)
  49. 淬火液(见配方)
  50. Mowiol ®(请参阅食谱)
  51. (可选)缓冲区A(请参阅配方)
  52. (可选)缓冲区B(请参阅配方)


  1. 微量离心机(例如,,Eppendorf,型号:5415或类似物)
  2. Nucleosil 100-5 C18色谱柱
  3. (可选)Dionex DNAPac PA200 4 x 250 mm色谱柱
  4. 血细胞计数器
  5. 细胞培养罩
  6. 热循环仪
  7. 铝金属板(长x宽x厚[cm]:例如,20×12×2)
  8. 细胞培养箱
  9. 半弯钳
  10. 烤箱
  11. STED显微镜,Leica脉冲STED设置由真实共聚焦系统(TCS)STED SP5(Leica Microsystems,型号:Leica TCS SP5)荧光显微镜组成,荧光显微镜配备有100x 1.4 NA HCX PL APO油物镜(Leica Microsystems,德国) >
  12. 脉冲激光(PicoQuant,德国)
  13. 蓝宝石可调激光(迈泰宽带,光谱物理,美国)
  14. 玻璃烧杯
  15. 磁力搅拌器
  16. 实验室外套,眼睛护理


  1. ImageJ( http://imagej.nih.gov/ij/ docs / index.html
  2. MATLAB(MathWorks,马萨诸塞州,美国)


  1. 染料与适体偶联
    在该步骤期间,进行硫醇 - 马来酰亚胺交联反应以使荧光团与适配体缀合。或者具有3'-氨基的适体可用于与NHS酯(例如,Atto647N NHS-酯)的进一步缀合。一些荧光标记的适体也可商购。
    1. 在100μl0.1M TEAA中加入10nmol硫醇的适体与10mM Tris(2-羧乙基)膦溶液(TCEP;参见方案1)。将管在70℃加热3分钟,并将混合物在室温下孵育1小时
    2. 根据制造商的说明书,在Biospin 6柱上通过尺寸排阻色谱法除去此反应。
    3. 加入还原适体(来自步骤A2)将4μl10μg/μl马来酰亚胺官能化的Atto647N(预先溶于DMSO中),并通过移液或涡旋混合内容物。
    4. 在4℃下孵育过夜。
    5. 通过乙醇沉淀回收荧光团标记的适体:加入100μl300mM NaCl和450μl冷乙醇,通过倒置封闭样品几次仔细混合,并将样品在干冰中冷冻20分钟(或-20℃,数小时),在4℃下以15,000 xg(rcf)离心30分钟,弃去上清液,并将沉淀物在50μl的70%乙醇中洗涤。
    6. 重悬于50μl1x DPBS中,并在Biospin 6柱上脱盐1次DPBS
    7. 测量260 nm和650 nm的吸光度比
    8. 在Nucleosil 100-5 C18 4 x 250 mm柱上使用0-40%乙腈在TEAA缓冲液中的梯度在30分钟内通过反相HLPC(例如)确认标记效率和游离染料不存在在30℃下的流速为1ml / min)或通过阴离子交换HPLC(例如,使用0-75%缓冲液B的梯度的Dionex DNAPac PA200 4×250mm柱)(参见配方12)在40分钟的缓冲液A(参见食谱11)中,流速为1ml / min,在60℃。
    9. 将标记的适体样品储存在-20°C直至使用。

  2. 染色前的细胞培养准备 含有感兴趣受体的细胞系(和缺乏它的细胞系,用作阴性对照)应在适配体染色前一天接种到含有聚-L-赖氨酸(PLL)的包被盖玻片的12孔板中。根据细胞系,选择适当的细胞编号(使用血细胞计数器计数细胞),在12-16小时孵育后达到约70-80%汇合。下面描述的整个过程必须在细胞培养罩下的无菌条件下进行。
    1. 将HeLa(Epha2a阳性)细胞在完全DMEM培养基(参见方案2)和A-431(EGFR阳性)和SKBR3(ErbB2R阳性)细胞在完全RPMI培养基中培养(参见方案3)生长在平板中。所有细胞系保持在37℃和5%CO 2
    2. 为了分裂细胞,首先用无菌的1x DPBS洗涤细胞,加入2-3ml胰蛋白酶-EDTA以覆盖板的表面,并在37℃下孵育板1-5分钟或直到细胞完全分离。加入10毫升完整的DMEM培养基或RPMI培养基以灭活胰蛋白酶。轻轻吸取含有灭活胰蛋白酶的培养基,将所有细胞从板上分离,并将其转移到无菌的15 ml管中。
    3. 在室温下以1,000rpm(〜250×g×g)离心细胞4分钟。
    4. 吸出上清液并将细胞沉淀重悬于10ml完全DMEM培养基/ RPMI培养基中。在新鲜的完全DMEM或RPMI培养基中稀释悬浮的细胞至所需浓度,并向含有PLL处理的盖玻片的12孔板的每个孔中加入1ml(用于PLL储备溶液制备和稀释,参见方案4)。将板在37℃和5%CO 2孵育直到染色。

  3. 功能适体的制备:折叠反应
    在该步骤中,适体以适当的镁浓度暴露于高温,这允许它们在染色程序之前获得适当的折叠。为了获得更好的效果,折叠反应应在染色前每次新鲜进行。在开始折叠反应之前,准备在1x DPBS中稀释适体的储备溶液至终浓度为30μM,并保存在4°C的光照条件下。
    1. 通过混合3.3μl荧光标记的适体(30μM适体储备溶液),2μl5x PBS(参见方案5),2μl5x MgCl 2(2),制备10μl10μM功能适体食谱6)和2.7μl超纯DNA酶和无RNA酶的蒸馏水。请注意,该反应应在适合热循环仪的PCR管中进行。
    2. 将适体溶液加热至75℃3分钟,然后使用热循环仪以1℃/ min的速率冷却至20℃。

  4. 适体活细胞染色
    1. 从程序B中制备的细胞吸出培养基,根据细胞系,用完全的DMEM或RPMI冲洗一次,并在37℃和5%CO 2(细胞培养物)中孵育10分钟 培养箱),500μl/孔新鲜制备的含有剪切的鲑鱼精子DNA和硫酸葡聚糖的封闭溶液(参见方案7)。
    2. 同时,准备用于37℃染色/培养的金属板。 用胶带固定一块Parafilm ®(足够大以适合所有盖玻片被染色)(见视频1),并预热含有Parafilm ®的金属板, / sup> M在细胞培养箱中。

      Video 1. Aptamers live staining part I. This video shows the metal plate preparation for aptamer live staining. Please note that this video is for demonstration only and that this step should be performed inside a cell culture hood.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

    3. 封闭后(步骤D1),使用半弯钳剔除12孔板上的盖玻片,用柔软的纸巾轻轻敲打盖玻片的边缘,取出多余的封闭溶液,将盖玻片倒置在60 点样固定在金属板(步骤D2中制成)的Parafilm固定体上(参见视频)(见视频),将染色溶液(完全补充有100μg/ ml剪切的鲑鱼精子DNA和250nM折叠适体的DMEM)2)。 在37℃和5%CO 2下孵育细胞60分钟。

      Video 2. Aptamers live staining part II. This video shows removal of coverslips containing cells from the 12-well plates using half-curved-forceps and incubation of cells (on coverslips) with staining solution. Please note that this video is for demonstration only and that this step should be performed inside the cell culture hood. To avoid contamination, gloves should be sprayed with 70% ethanol.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

    4. 孵化后,用半弯钳仔细将盖玻片从金属板上取下,用柔软的纸巾轻轻敲打盖玻片的边缘去除多余的染色溶液,然后大量淹没数次(< 例如,20-40ml在一个小烧杯中)的冰冷的1×DPBS。 通过将它们轻敲掉在薄纸上,并将每一个(细胞侧向上)放入装有1ml /孔冰冷的4%PFA的新的12孔板的孔中,从盖玻片上简单地除去多余的1x DPBS( 见配方8)进行固定(见视频3)。

      Video 3. Aptamers live staining part III. This video shows the washing step after staining procedure. Please note that video is in open air for demonstration only and that 4% PFA should only be handled in the fume hood.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

    5. 在冰上将细胞固定20分钟,然后在室温下再静置25分钟。固定期间,保护板免受光照,避免荧光团漂白
    6. 吸出PFA溶液,并向每个孔中加入1ml淬灭溶液(参见食谱9)。在室温下孵育15分钟,防止光照。
    7. 用1x DPBS(1ml /孔)洗涤两次5分钟,并用Mowiol ®(参见配方10;例如)安装盖玻片,8-9μl,用于18mm盖玻片)在显微镜玻璃片上
    8. 将安装的盖玻片在37℃的烘箱中干燥20-30分钟或在室温过夜,并保存在4℃,免受光照直至成像。


成像:使用100x 1.4 NA HCX PL APO油物镜获取本协议(图1)所示的图像。用635nm脉冲激光(PicoQuant,Germany)激发Atto647N荧光团,并用脉冲红外钛:蓝宝石可调激光器(Mai Tai Broadband,Spectra-Physics,USA)获得750nm消耗的STED光束。像素大小设置为20.2nm,扫描速度为1kHz,线平均为96次,针孔为1个艾里单位,信号用雪崩光电二极管检测器(APD)检测。

图1.适体或抗体染色细胞中的内体样结构的识别A.A STED图像比较用适体和抗体染色的细胞与相同受体(Ab1和Ab2)在饱和条件下,如(Gomes de Castro等人,2017)。箭头指向一些内体样结构的实例,使用抗体进行染色导致细胞器轮廓的不连续标记(由STED图像中的黄色圆圈表示) )该方案代表我们目前的假设,表明大的亲和力探针(如上图)可能不能检测到所有可用的表位,小分子如适体(下图)更好地装饰目标结构。


  1. 确定感兴趣的适体的饱和浓度或最佳染色浓度(最佳信噪比),固定染色的孵育时间(例如,60分钟),并测试不同的适体浓度范围为10推荐阳性细胞系中-500nM(或更多)。用不同浓度的适体进行染色,固定和固定后,细胞可以在任何荧光显微镜中成像,荧光强度可以使用ImageJ或MATLAB进行计算。
  2. 在定义染色程序的饱和条件后,下一步是测试结合特异性。这是通过评估表达受体的细胞和不表达受体的细胞的染色来实现的。重要的是,阳性细胞和阴性细胞的染色条件,适体浓度和孵育时间必须相同。一旦染色和固定,细胞可以用落射荧光或共焦显微镜成像。阴性细胞系几乎不显示特定的荧光信号。为了适当控制非特异性染色背景,可以平行测定与与特异性靶标结合的适体结合的相同荧光团的随机适体的免疫标记。此控制不应有任何荧光信号。另外的对照是通过使用相同对照的荧光流式细胞术分析来评估结合特异性
  3. 每个受体都有自己的内化动力学。在这项工作和协议中,我们打算通过内部化来最大化标签,从而选择更长的孵化时间。
  4. 如果适体含有BzdU(5-(N-苄基羧酰胺)-2'-脱氧尿苷)或任何其他疏水基团,我们建议向阻断溶液中加入硫酸葡聚糖。阻塞溶液中硫酸葡聚糖浓度的优化强烈地降低了由适配体与PLL处理的盖玻片的静电相互作用引起的非特异性结合引起的背景。
  5. 如果适配体的非特异性结合仍然存在,则在染色溶液中增加剪切的鲑鱼精子DNA和/或硫酸葡聚糖的浓度。聚阴离子竞争剂/封闭剂硫酸葡聚糖还基本上减少由聚阴离子适体的静电吸引引起的非特异性结合到核中的带正电荷的位点,例如组蛋白。
  6. 为了鉴定和定量内质体样结构,强烈推荐共定位研究(Gomes de Castro等人,2017)。


  1. 1M Tris(2-羧乙基)膦盐酸盐储备溶液(TCEP)
    100 ml:
    1. 加入11.47克TCEP至35毫升冷超纯水
    2. 用10N NaOH将pH调至7.0,并将体积调整至100ml
    3. 等分成2.0毫升管,储存于-20°C
  2. 完成DMEM培养基
    1. 补充有10%FBS,4mM L-谷氨酰胺和100U / ml青霉素和链霉素的DMEM
    2. 通过真空过滤灭菌
    3. 储存于4°C
  3. 完成RPMI培养基
    1. 补充有10%FBS,4mM L-谷氨酰胺和100U / ml青霉素和链霉素的RPMI
    2. 通过真空过滤灭菌
    3. 储存于4°C
  4. 聚-L-赖氨酸(PLL)储备溶液
    1. 在超纯水中制备2 mg / ml储备溶液,并通过0.2μm的注射器过滤器进行消毒
    2. 将等分试样储存在-20°C
    3. 在无菌罩下准备含有PLL处理的盖玻片的板材
    4. 向含有盖玻片的每个孔中加入1毫升0.1毫克/毫升的PLL
    5. 在室温下孵育1小时
    6. 孵育后,用超纯水洗涤两次,并将板在细胞培养罩内空气干燥
    7. 将板储存在4°C直到使用
  5. 5倍磷酸缓冲盐水(5倍PBS)
    1. 在超纯水中稀释10倍浓缩的PBS储液。用0.2μm注射器过滤器或高压釜过滤细胞培养罩内的溶液。存储在RT
    2. 对于1 L 10x浓缩的PBS储备溶液:
      溶于DNase和RNase-free水中 将pH调节至7.4
  6. 25mM MgCl 2溶液(5×MgCl 2)
    1. 在无DNA酶和无RNase的蒸馏水中将1M MgCl 2/2储备液稀释至25mM。为了避免颗粒和污染,请用0.2μm注射器过滤器过滤细胞培养罩内的溶液。在室温下存放
    2. 对于100ml的1M MgCl 2 储备溶液:
      将20.3g MgCl 2·6H 2 O溶于70ml DNase和RNase-free水中,并将体积调节至100ml
  7. 阻塞解决方案
    补充有100μg/ ml剪切鲑鱼精子DNA和1mM硫酸葡聚糖硫酸盐的DMEM或RPMI完全培养基
  8. 4%多聚甲醛(PFA)
    1升PFA 4%制剂:
    1. 在玻璃烧杯中加入约600ml的1x PBS至40g的PFA,并使用磁力搅拌器在〜50℃下搅拌。
    2. 将pH调节至7到8之间
    3. 用1x PBS将体积调节至1L,使其等分并储存在-20°C
  9. 淬火液
    1. 0.1M甘氨酸在1x DPBS中
    2. 准备100ml甘氨酸1M储备溶液:
      将7.5 g甘氨酸溶于超纯水中 通过0.2μm注射器过滤器过滤,并在RT存储下
  10. 的Mowiol ®
    1. 混合24g甘油,9.6g Mowiol,4-88试剂,62.4ml蒸馏水和9.6ml 1M Tris缓冲液,圆锥形圆筒,磁力搅拌器5-7天
    2. 任选地在40-50℃下加热混合物以溶解Mowiol
    3. 沉淀后,将上清液以等分试样(例如,2.0ml管)分离,并在4℃下储存
  11. (可选)缓冲区A
    25mM Tris-HCl,pH8
    6 M尿素
  12. (可选)缓冲区B
    在25mM Tris-HCl pH8,6M尿素中的0.5M NaClO 4


这里描述的该协议更详细地已经在(Gomes de Castro等人,2017)中公开。这项工作得到了杰出团队和DFG研究中心纳米显微镜和脑分子生理学(CNMPB)的支持。


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引用:Gomes de Castro, M. A., Höbartner, C. and Opazo, F. (2017). Staining of Membrane Receptors with Fluorescently-labeled DNA Aptamers for Super-resolution Imaging. Bio-protocol 7(17): e2541. DOI: 10.21769/BioProtoc.2541.