Labeling Postsynaptic Densities for Super-Resolution Microscopy With Minimal Signal-Loss and Offset

Materials and reagents

Biological materials

1. P0–P1 mouse, C57BL/6J strain

Reagents

1. Sylites (iSylite: Nanotag, catalog number: P4001; eSylites and a range of conjugates are also available via mail to hans.maric@uni-wuerzburg.de and jwhell@ucdavis.edu

2. Poly-D-lysine (PDL) hydrobromide (Sigma-Aldrich, catalog number: P6407-5MG, CAS number: 27964-99-4)

3. UltraPure^TM DNase/RNase-free distilled water (Thermo Fisher, catalog number: 10977015)

4. Gibco^TM HBSS, no calcium, no magnesium (Thermo Fisher, catalog number: 14170112)

5. Gibco^TM HBSS, calcium, magnesium (Thermo Fisher, catalog number: 24020117)

6. B-27^TM Plus Supplement (50×) (Thermo Fisher, catalog number: A3582801)

7. Neurobasal^TM Plus medium (Thermo Fisher, catalog number: A3582901)

8. GlutaMAX^TM supplement (Thermo Fisher, catalog number: 35050061)

9. Trypsin-EDTA (0.05%), phenol red (Thermo Fisher, catalog number: 25300054)

10. Gentamicin solution (Sigma-Aldrich, catalog number: G1397-10ML)

11. PBS, pH 7.4 (Thermo Fisher, catalog number: 10010023)

12. Pierce^TM 16% formaldehyde (w/v), methanol-free (Thermo Fisher, catalog number: 28906)

13. Glyoxal 40 wt. % solution in water (Sigma-Aldrich, catalog number: 128465)

14. Fish serum blocking buffer (Thermo Fisher, catalog number: 37527)

15. Triton X-100 (Sigma-Aldrich, catalog number: T9284-100ML, CAS number: 9036-19-5)

16. ProLong^TM Gold antifade mountant (Thermo Fisher, catalog number: P10144)

17. Paraformaldehyde, 16% w/v aqueous solution, methanol-free (Thermo Fisher, catalog number: 043368.9M)

18. Ethyl alcohol, pure (Sigma-Aldrich, catalog number: 459836-100ML)

19. Acetic acid, glacial (Sigma-Aldrich, catalog number: 695092-100ML)

20. NaOH (Fisher Scientific, catalog number: S318)

Solutions

1. PDL solution (see Recipes)

2. Culturing medium (see Recipes)

3. 4% paraformaldehyde (PFA) solution (see Recipes)

4. Glyoxal buffer (see Recipes)

5. Blocking and permeabilization buffer (see Recipes)

Recipes

1. PDL solution

5 mg of Poly-D-lysine hydrobromide in 50 mL of ultrapure water.

2. Culturing medium

Prepare and equilibrate in a CO₂ incubator one day prior to dissection.

3. 4% PFA solution

1 mL of 16% PFA in 3 mL of PBS, pH 7.4.

4. Glyoxal buffer

Add 0.856 mL of 40 wt. % glyoxal solution to 10 mL of glyoxal buffer prior to fixation [11].

5. Blocking and permeabilization buffer

For surface labeling with antibody, block without adding Triton X-100 and permeabilize after surface labeling.

Blocking is primarily for antibody co-staining. When using Sylites alone, serum (either fish serum or 3% BSA) can be omitted without introducing nonspecific labeling.

Laboratory supplies

1. Round coverslips, #1.5 18 mm (Warner Instrument, catalog number: 64-0714)

2. Microscope slides (Globe Scientific, catalog number: 1338)

3. 12-well plate (Greiner, catalog number: 665180)

4. T25 flask/50 mL conical tube (Corning, catalog number: 430639)

5. Straight forceps

6. 4” micro straight scissors, Castroviejo stainless steel (Electron Microscopy Sciences, catalog number: 72933)

8. Freer elevator/spatula

9. 60 mm Petri dish (Celltreat, catalog number: 229663)

10. Scalpel

11. Ultra-fine forceps (Excelta Corp, model: 7-SA)

12. Pipette tips

13. Parafilm

14. 150 mm Petri dish (Celltreat Scientific, catalog number: 229656)

Equipment

1. CO₂ incubator (Heraeus HERAcell 240 CO₂ Incubator)

2. Dissection microscope (AmScope, model: SM-1)

3. Spectrophotometer/NanoDrop (BioTek Synergy 2 with Take3 plate)

3. Fluorescence microscope (Leica, model: TCS SP8 gated STED 3× microscope)

Procedure

A. Preparation of dissociated hippocampal neurons on coverslips

Note: All procedures involving animals followed the guidelines for the Care and Use of Laboratory Animals from the US National Institutes of Health and were approved by the Institutional Animal Care and Use committees at the University of California, Davis. If you wish to prepare the primary neuronal cultures that are part of this protocol, ensure that you have all institutional permissions from the appropriate authorities for the use of the respective laboratory animals.

1. One day prior to P0–P1 mouse dissection, prepare coated coverslips by putting 18 mm #1.5 coverslips in individual wells of a 12-well plate.

2. Add 270 μL of PDL solution on top of the coverslips. Ensure that the solution forms a stable meniscus on top of the coverslip without spilling over to the bottom of the well, as shown in Figure 1.

Figure 1. Poly-D-lysine (PDL) coating on coverslips. Limiting PDL to the coverslip surface restricts cells to the coverslip during seeding, preventing them from spreading to the bottom of the plate.

3. Incubate the 12-well plate overnight at 4 °C.

4. Prepare culturing medium in a 50 mL T25 flask with a vented cap or in a 50 mL conical tube with the cap loosely closed. Incubate in a CO₂ incubator overnight at 37 °C.

5. Before dissection, rinse the coverslips three times with ultrapure water and allow them to air-dry inside the biosafety cabinet with the lid removed. Air drying usually takes approximately 1 h and is normally completed by the time the dissection steps are finished.

6. Decapitate E18 or P0–P1 pups at the base of the skull, then stabilize the head by inserting a pair of thin straight forceps into the eye sockets (Video 1).

7. Cut the skin along the sagittal suture from the posterior to the anterior side with 3.5 mm Castroviejo micro scissors.

8. Cut the skull along the sagittal suture from the posterior to the anterior side and along the bone plate below the ears on both sides.

9. Carefully scoop out the brain by using a freer elevator or a spatula and immerse it in precooled HBSS solution without calcium and magnesium in a 60 mm Petri dish.

10. Slice off the cortex from the midbrain by using a scalpel.

11. Using a dissection microscope, carefully peel off the meninges with ultrafine forceps (Video 2).

12. Orient the hemisphere with the dorsal side of the brain facing upward. The hippocampi should be clearly visible.

13. Remove the hippocampi from the cortex by using forceps. For detailed procedures and pictures, see [12].

14. Transfer the isolated hippocampi into a 5 mL conical tube containing fresh HBSS without Ca²⁺ and Mg²⁺ solution and keep on ice while dissecting the remaining animals (two pups are sufficient to prepare one 12-well plate).

15. Remove the HBSS solution and rinse with prewarmed 0.05% trypsin-EDTA solution twice.

16. Incubate the tissue in 5 mL of fresh, prewarmed 0.05% trypsin-EDTA solution at 37 °C for 15 min, gently flipping the tube midway through the incubation.

17. Remove the trypsin-EDTA solution, then quench any residual enzymatic activity by washing the tissue three times with HBSS containing calcium and magnesium.

18. Replace the solution with 1 mL of pre-equilibrated culture medium.

19. Carefully triturate the tissue using a 1 mL pipette tip trimmed to different pore sizes: 11 times with a large-pore tip, 11 times with a medium-pore tip, and 11 times with an uncut tip.

20. Allow the undissociated tissue chunks to settle by gravity for 1 min.

21. Take out the supernatant and perform a cell count using a hemocytometer with a 5–10-fold dilution.

22. Dilute cell suspension to a final density of 360,000 cells/mL with pre-equilibrated culture medium.

23. Add 250 μL of the suspended cells on top of the pre-coated coverslip. The solution should form a stable meniscus on top of the coverslip without spilling over to the bottom of the well.

24. Incubate the cells in a CO₂ incubator for at least 2 h for the cells to adhere onto the coverslip.

25. Add 750 μL of culture medium to make up the volume to 1 mL.

26. Incubate the cell in a CO₂ incubator for at least 14 days and refresh 40% of the culture medium every 7 days.

B. Neuron fixation

1. Fix the neurons at the desired days in vitro (DIV) by first removing the culture medium and rinsing twice with PBS, followed by a 15-min incubation with either 4% PFA or glyoxal solution. Ensure that cells do not dry out during solution changes. While glyoxal fixation may offer improved image quality, no significant difference in labeling efficiency has been observed between PFA and glyoxal.

2. Remove fixation solution and gently wash the cells with PBS for 5 min on an orbital shaker, repeating the wash three times.

Pause point: Fixed neurons can be stored at 4 °C after fixation for up to 2 weeks.

Critical: Sylite labeling is sensitive toward the choice of fixation reagent. eSylite labeling is sensitive to the integrity of the PDZ binding pockets. Cells stored for more than one month may exhibit collapsed binding pockets, resulting in a loss of eSylite signal, while PSD-95 antibody staining may still be detectable.

Caution: Glyoxal and PFA are environmentally hazardous. Quench excess aldehydes by incubating with 1 M glycine for 15 min before discarding the solution down the drain.

C. Sylite labeling

1. Block and permeabilize the cells using a blocking/permeabilization solution (see Recipe 5) for at least 30 min at room temperature. Cells can be kept in the blocking/permeabilization solution for 1–2 days at 4 °C.

Note: For extracellular labeling, perform blocking and permeabilization separately, omitting Triton X-100 during the blocking step. Fish serum or 3% BSA in PBS can be used as the blocking reagent. Blocking is not necessary when using Sylites alone and is primarily required when additional antibodies are included in the labeling cocktail.

2. Dilute the Sylite solution to a final concentration of 25–100 nM in blocking and permeabilization solution. While labeling is possible at concentrations as low as 1 nM, lower concentrations may result in incomplete labeling and reduced signal intensity. Additional antibodies can be included in the same solution for multichannel imaging.

3. Add 100 μL of the Sylite-containing blocking and permeabilization solution onto a flat sheet of Parafilm placed in a 150 mm Petri dish.

4. Carefully pick up the coverslips with forceps, flip them, and place them cell-side down onto the Sylite solution for incubation. Incubate for at least 30 min at room temperature or overnight at 4 °C. Incubation for 30 min or 2 h at room temperature did not show a discernible difference under diffraction-limited imaging; however, 1 h at room temperature is recommended to ensure efficient co-labeling with antibodies in the cocktail. Avoid exposing the samples to strong light after Sylite labeling. For overnight incubation, place a wet Kimwipe inside the Petri dish to maintain humidity and prevent the solution from drying out.

5. Transfer and flip the coverslip back to a 12-well plate. Gently wash the cells with PBS for 5 min on an orbital shaker; repeat the wash three times.

6. Secondary antibody labeling can be performed at this step, if needed.

D. Imaging with fluorescence microscope

1. For confocal/STED microscopy, add one drop of ProLong^TM Gold antifade mountant onto a clean microscope slide. Pick up the coverslip and gently blot the edge with a Kimwipe to remove excess PBS. Carefully place the coverslip onto the mountant, cell-side down, and avoid trapping air bubbles during mounting.

Critical: Sylite with sulfoCy5 labeled, designed for dSTORM, is not photostable enough even with the mountant. Imaging with the sulfoCy5 version of Sylite should be done with less laser power and multiple line average and frame accumulation to increase the signal-to-noise (S/N) ratio and signal intensity. The STAR635P version of Sylite is photostable and easier to image with confocal and STED microscopy.

2. Allow the mountant to cure at room temperature for 24 h, keeping the slide protected from light during this period.

3. Rinse the coverslip surface with ultrapure water and gently blot dry to remove any residual PBS salts, which can damage the objective lens.

Pause point: Mounted coverslips can be stored at 4 °C in the dark for several weeks without significant signal loss.

4. Proceed with imaging according to the specific instructions for your microscope system.

Validation of protocol

Images of Sylites applications in fixed primary neuron imaging are shown in Figure 2.

Figure 2. Image gallery of Sylite probes. (A) Confocal imaging of eSylite (sCy5-labeled) in 23 days in vitro (DIV) dissociated neuronal cultures, co-labeled with antibodies against MAP2, GluA2, and GluN2B. (B) tau-STED imaging of STAR635P-labeled eSylite in eGFP-transfected dissociated 21 DIV cortical neurons, co-labeled with a PSD-95 antibody visualized by Alexa Fluor 594-conjugated secondary antibody. Cumulative frequency distributions of FWHM of tau-STED-resolved nanoclusters labeled by the antibody (cyan line, n = 236 nanoclusters) and eSylite (magenta line, n = 239 nanoclusters; ***p < 0.0001, K−S test). Reprinted (adapted) with permission from [1]. (C) Confocal imaging of iSylite (sCy5-labeled) in 20 DIV dissociated neuronal cultures, co-labeled with antibodies against VGAT and VGlut1.

This protocol or parts of it has been used and validated in the following research article:

1. Huhn et al. [1]. eSylites: Synthetic Probes for Visualization and Topographic Mapping of Single Excitatory Synapses. Journal of the American Chemical Society.

2. Khayenko et al. [2]. A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity. Angewandte Chemie International Edition.

General notes and troubleshooting

General notes

1. Cell culture conditions and seeding density significantly influence neuronal development and final image quality. As a general guideline, seed approximately 40,000 cells on 12 mm coverslips, 90,000 cells on 18 mm coverslips, and 200,000 cells on 25 mm coverslips. Neurons older than 25 DIV may begin to degenerate, often exhibiting segmented dendrites with bubble-like structures.

2. The stock concentration of Sylite should be calibrated using a UV/Vis spectrophotometer or a NanoDrop^TM instrument, based on its absorption maximum. Working concentration of Sylites is optimal within 5−100 nM; low concentrations result in insufficient labeling, while high concentrations, up to 1 μM, generally have no effect but might cause higher background if washing is incomplete.

3. dSTORM imaging using sulfo-Cy5-labeled Sylite is not strongly recommended due to its extreme sensitivity to photobleaching. Significant loss of localizations can occur even during focus adjustment and TIRF angle alignment under weak laser illumination, despite the presence of Trolox, PCA, and PCD. This makes imaging conditions highly finicky and unreliable.

Troubleshooting

Problem 1: No staining was observed.

Possible cause: Suboptimal cell culture conditions or prolonged post-fixation storage.

Solution: Co-label with a synaptic marker antibody to confirm the health and synaptic maturity of the neurons. Perform Sylite labeling immediately after fixation to ensure the integrity of the unoccupied binding pocket. Note that hippocampal neurons are predominantly glutamatergic pyramidal neurons, while GABAergic interneurons represent a smaller population. In dissociated neuronal cultures, different cell types may localize unevenly, which can result in the absence of detectable excitatory or inhibitory synapses in certain areas. Make sure to scan over different fields in case of an inhomogeneous cell type distribution.

Problem 2: Weak and noisy image quality.

Possible cause: Suboptimal image acquisition condition.

Solution: For high noise, apply frame averaging to improve the signal-to-noise (S/N) ratio. For a weak signal, increase laser power, adjust detector gain, or enable frame accumulation to enhance image brightness without compromising resolution. If a strong reflection from the incident light is observed and cannot be eliminated by the filter cube, try applying time gating (~0.4–0.6 ns gate) if your imaging system supports it. This can effectively suppress short lifetime autofluorescence and reflected excitation light, improving image contrast.

Acknowledgments

The work shown in this protocol was published in Journal of the American Chemical Society [1] and Angewandte Chemie International Edition [2]. H.M.M. and J.W.H. contributed to the experiment’s conceptualization and investigation. S.H., C.H., and A.S. carried out experiments. S.H. wrote the original draft. J.W.H. and H.M.M. reviewed and edited the final version. M.H. was supported by NIH (20GM109098), the National Science Foundation (2242771), and the Alzheimer’s Association (AARG-NTF-231150820). H.M.M. acknowledges support from the Junior Group Leader program of the Rudolf Virchow Center, the excellent ideas program of the University of Würzburg (JMU), and the Emmy Noether Program of the Deutsche Forschungsgemeinschaft (DFG) (MA6957/1-1). J.W.H. was supported by a WM Keck award and NIH grant RF1 AG055357.

Competing interests

The authors declare the following competing financial interest(s): H.M.M. and C.H. hold utility models on Sylites.

References

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