3D STED Super-Resolution Imaging Strategy for Visualizing Synaptic Nano-architecture in Brain Cryosections
用于脑组织冰冻切片突触纳米结构可视化的三维STED超分辨成像策略
(*Contributed equally to this work, §Technical contact: js0232@mix.wvu.edu; adam.skeens@hsc.wvu.edu) 发布: 2026年04月05日第16卷第7期 DOI: 10.21769/BioProtoc.5644 浏览次数: 659
评审: Munenori IshibashiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2025
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Abstract
Super-resolution imaging of synapses in intact brain tissue remains challenging because light scattering, photobleaching, and limited probe penetration, along with antigen accessibility within the densely packed postsynaptic densities (PSDs), constrain resolution and labeling efficiency. Here, we present a protocol utilizing thin brain cryosections and tau-stimulated emission depletion (STED) nanoscopy to visualize the intricate nano-architecture of excitatory synapses in situ. Slicing the brain into 6 μm sections allows for highly efficient and even penetration of probes throughout sections while ensuring that the resolution is not significantly impacted by the imaging depth of the tissue. We outline step-by-step instructions for labeling pre- and postsynaptic nano-architecture using antibodies and nanobodies, highlighting how fixative choice influences the labeling efficiency of synaptic proteins. While this protocol is compatible with both confocal and super-resolution imaging, when combined with rapid image acquisition times of tau-STED, it enables clear separation of key synaptic features in three dimensions with minimal photobleaching. Thus, this approach enables robust multiplex imaging of fluorescently labeled synaptic proteins in the brain, providing exceptional spatial resolution for visualization and quantification of synaptic nanoarchitecture in its native environment.
Key features
• Detailed protocol for in situ 3D STED microscopy with ~50 nm XY and ~100 nm Z resolution.
• Optimized strategies for labeling pre- and postsynaptic nano-architecture using antibodies and nanobodies, including guidance on fixative choice.
• Unified workflow for visualizing synaptic morphology and nanoarchitecture to uncover molecular synaptic diversity in the brain at the nanoscale.
Keywords: Tau-STED microscopy (Tau-STED显微技术)
Brain cryosections (脑组织冰冻切片)
PSD-95 (PSD-95)
Excitatory synapse (兴奋性突触)
Nanobody (纳米抗体)
Paraformaldehyde (多聚甲醛)
Glyoxal (乙二醛)
Graphical overview
Graphical overview of the procedure. Schematic outlining each step of the protocol from day 1 to day 15. The timeline for each step is indicated. The protocol is divided into four parts: (1) Perfusion/fixation, (2) cryosectioning, (3) immunolabeling, and (4) imaging, each described in detail in the text below. The entire workflow takes approximately two weeks to complete and includes multiple pause points (red circles) where the procedure can be temporarily paused or where brain tissue can be safely stored.
Background
Over the last decade, the development of diverse super-resolution imaging platforms has enabled the investigation of cell-biological questions with unprecedented detail. For the field of neuroscience and synaptic biology, in particular, super-resolution microscopy transformed our understanding of the molecular mechanisms that underlie synaptic transmission and plasticity—the fundamental processes that are key for learning and memory [1,2]. Yet, despite these advances, the methodology has been limited mainly to studies in primary neurons from the cortex and hippocampus [3–11]. However, the brain contains billions of neurons and trillions of synapses, organized into distinct anatomical layers and domains that encompass a vast diversity of synaptic subtypes with unique molecular, structural, and functional characteristics [12–16]. Therefore, achieving transformative discoveries in the molecular biology of synapses, in both health and disease, will require super-resolution imaging approaches that capture the nanoscale organization of synaptic proteins directly within their native brain environment [17,18].
Immunolabeling offers a robust way to examine the molecular complexity of synapses in the brain [19-21]. Yet, several challenges hinder the application of super-resolution microscopy in situ. First, the intricacies of brain tissue, with its high lipid content and dense neuropil, make it difficult to resolve molecules in sub-diffraction structures such as synapses with high precision [22,23]. Critically, resolution decreases with tissue depth [24]. Thus, combined with the limited penetration of large probes to the depths of brain tissue, this crucially limits super-resolution imaging in brain sections. Second, the postsynaptic densities (PSDs) of excitatory synapses are densely packed with thousands of distinct proteins, making molecules localized to this sub-structure notoriously hard to label with conventional reagents [25,26]. Third, paraformaldehyde fixation is known to mask antigens due to the high degree of cross-linking, further reducing probe affinity [27]. Altogether, these limitations reduce labeling efficiency, compromise the signal-to-noise (S/N) ratio, and leave weaker signals more susceptible to photobleaching.
In this protocol, we describe strategies to overcome these limitations by providing detailed instructions on how to achieve highly efficient immunolabeling of synapses in brain cryosections, thereby maximizing the ability to robustly identify synaptic nano-architecture in situ in three dimensions. The full workflow takes approximately two weeks and contains several steps, such as brain isolation, preparation of thin cryosections, and immunolabeling. Our primary focus is to provide guidance on tissue preparation, including the choice of fixative, and on selecting probes for efficient labeling of proteins in pre- and postsynaptic terminals. To this end, we highlight the use of small, single-domain nanobodies as an alternative to larger conventional antibodies, enabling highly multiplexed, high-resolution imaging of molecular nano-organization of synapses in brain tissue. We then describe the experimental steps and imaging configuration required to visualize synaptic nano-architecture in labeled brain sections using three-dimensional stimulated emission depletion (3D-STED) microscopy, with particular emphasis on the fluorescence lifetime imaging (FLIM)-based Leica Stellaris STED platform. Importantly, this protocol can be readily adapted to other super-resolution imaging approaches, including expansion microscopy [28]. Together, this protocol provides a unified framework for immunolabeling-based imaging of synaptic nano-architecture in situ.
Materials and reagents
Biological materials
1. Mouse: B6. Cg-Tg (Thy1-YFP)16Jrs/J (Jackson Laboratory, Strain: 003782/ RRID: IMSR_JAX:003782) [29]
Reagents
1. 2-Methylbutane (Fisher Scientific, catalog number: O3551-4)
2. Acetic acid (Fisher Scientific, catalog number: A38S-500)
3. Antibodies and nanobodies (various suppliers, see Table S1)
4. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: BP510-500)
5. Chromium (III) potassium sulfate (Sigma-Aldrich, catalog number: 60152-100G)
6. D-(+)-Glucose (Sigma-Aldrich, catalog number: G7021-1KG)
7. D-Sucrose (Fisher Scientific, catalog number: BP220-1)
8. EM-grade paraformaldehyde (PFA) (Polysciences, catalog number: 00380-1)
9. Ethanol 95% (v/v) (Decon Labs, Inc., catalog number: 2801)
10. Gelatin from bovine skin (Sigma Aldrich, catalog number: G9391-100G)
11. Glyoxal 40% (w/v) solution in water (Sigma-Aldrich, catalog number: 128465)
12. Heparin (Akron Biotech, catalog number: AK3004-5000)
13. Isoflurane (Piramal Pharma Limited, catalog number: NDC66794-017-25)
14. Ketamine (Ketaset Injectable C IIIN, 100 mg/mL) (Patterson Veterinary, catalog number: 07-803-6637; Schedule III DEA license required)
15. Normal goat serum (Gibco, catalog number: 16210-064)
16. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: BP213-1)
17. PBS, pH 7.4 (Thermo Fisher, catalog number: 10010023)
18. Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366-500)
19. ProLongTM glass antifade mountant (Thermo Fisher, catalog number: P36984)
20. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761-500G)
21. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-500)
22. Sodium phosphate dibasic anhydrous (Na2HPO4) (Fisher Scientific, catalog number: BP332-500)
23. Sodium phosphate monobasic anhydrous (NaH2PO4) (Fisher Scientific, catalog number: BP329-1)
24. Tissue-Tek optimal cutting temperature (O.C.T) compound (Sakura FineTek USA, catalog number: 4583)
25. Triton X-100 (Sigma-Aldrich, catalog number: T9284-100ML)
26. Xylazine (AnaSed) injection 100 mg/mL (Patterson Veterinary, catalog number: 07-895-0792)
Solutions
1. Oxygenated artificial cerebrospinal fluid (ACSF), pH 7.4 (see Recipes)
2. 0.4 M phosphate buffer (PB) (see Recipes)
3. 4% (w/v) PFA solution, pH 7.4 (see Recipes)
4. 9% (v/v) glyoxal, 8% (v/v) acetic acid, pH 4 (see Recipes)
5. Gelatin solution (see Recipes)
6. Chromium potassium sulfate solution (see Recipes)
7. Blocking and permeabilization buffer (see Recipes)
Recipes
1. Oxygenated ACSF solution, pH 7.4
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| NaCl | 125 mM | 1.826 g |
| KCl | 2.5 mM | 0.046 g |
| CaCl2 (dihydrate) | 2 mM | 0.073 g |
| NaHCO3 | 25 mM | 0.525 g |
| NaH2PO4 | 1.25 mM | 0.037g |
| MgSO4 (heptahydrate) | 2 mM | 0.123 g |
| D-Glucose | 10 mM | 0.450 g |
| Heparin (191.5 U/mg) | 10 U/mL | 13 mg |
| Milli-Q H2O | Up to 250 mL | |
| Total | 250 mL |
Oxygenate the solution on ice for 30 min to 1 h immediately prior to use. Afterward, adjust to pH 7.4, if needed. Keep ice-cold. Add heparin right before use. Prepare the solution fresh each time.
2. 0.4 M PB
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Na2HPO4 | 320 mM | 45.68 g |
| NaH2PO4 | 87 mM | 10.49 g |
| Milli-Q H2O | Up to 1 L (800 mL before pH adjustment, rest after pH adjustment) | |
| Total | 1 L |
The solution should be at pH 7.4 when prepared, and no pH adjustment should be necessary. This stock solution is used to prepare 0.1 M PB by diluting it 1:4 in Milli-Q water for other solutions or washing steps in the protocol. The stock can be stored at room temperature (RT) for several months.
3. 4% (w/v) PFA solution, pH 7.4
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| EM-grade PFA | 4% (w/v) | 20 g |
| 0.4 M PB (Recipe 2) | 0.1 M | 125 mL |
| Milli-Q H2O | Up to 500 mL (300 mL before pH adjustment, rest after pH adjustment) | |
| Total | 500 mL |
Heat approximately 300 mL of Milli-Q H2O to ~60 °C in a 500 mL beaker on a heated stir plate in a fume hood (do not exceed 65 °C). Once the water reaches temperature, add the PFA and allow it to dissolve while stirring for approximately 30 min; the solution will remain slightly cloudy at this stage. Slowly add 10 N NaOH dropwise (~10 drops) while stirring until the solution becomes completely clear. Add 125 mL of 0.4 M PB (Recipe 2) to the PFA solution and adjust the pH to 7.4 using a pH meter. Transfer the solution to a 500 mL 0.45 μm Nalgene rapid-flow filter and filter under vacuum. Bring the solution to a final volume of 500 mL with Milli-Q H2O and chill on ice. This solution should be prepared fresh before use and should be used ice cold.
4. 9% (v/v) glyoxal, 8% (v/v) acetic acid, pH 4.0 (adapted from [27])
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Glyoxal 40% (w/v) solution in water | 9% (v/v) | 112.5 mL |
| Acetic acid | 8% (v/v) | 40 mL |
| 0.1 M PB | Up to 500 mL (250 mL before pH adjustment, rest after pH adjustment) | |
| Total | 500 mL |
This solution is very acidic due to the presence of acetic acid. A large amount of strong base (10 N NaOH) is required to bring the pH to 4. Chill on ice before using for perfusion. Prepare fresh each time.
5. Gelatin solution
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Gelatin from bovine skin | 5 mg/mL | 4.5 g |
| Milli-Q H2O | Up to 900 mL | |
| Total | 900 mL |
Use a heated stir plate to fully dissolve gelatin into the solution. Prepare fresh right before use.
6. Chromium potassium sulfate solution
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Chromium potassium sulfate | 1 mM | 450 mg |
| Milli-Q H2O | Up to 30 mL | |
| Total | 30 mL |
This solution should be prepared fresh right before use.
7. Blocking and permeabilization buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1× PBS | 1× | 9 mL |
| Normal goat serum | 5% (v/v) | 0.5 mL |
| 10% (v/v) Triton X | 0.5% (v/v) | 0.5 mL |
| Total | 10 mL |
The solution can be stored at 4 °C for ~1 month.
Laboratory supplies
1. Thermometer (-80 °C) (Fisher Scientific, catalog number: 13201640)
2. #1.5 rectangular coverslips (0.16–0.19 mm thick) (Fisher Scientific, catalog number: 22266882), gelatin-subbed (see section D for subbing procedure)
3. 10 cm Petri dishes (Fisher Scientific, catalog number: FB0875712)
4. 21 G × 1 in. Precision glide needle (BD, catalog number: 305165)
5. 2 L glass beaker (Fisher Scientific, catalog number: FB101-2000)
6. 60 mL disposable syringe non-sterile catheter tip (BD, catalog number: 301037)
7. Aluminum foil roll (FisherBrand, catalog number: 01-213-103)
8. Cotton ball McKesson medium non-sterile (Mckesson Medical-Surgical, catalog number: 980221)
9. CoverWell incubation chambers 22 mm × 40 mm × 0.2 mm (GraceBio, catalog number: 645402)
10. Disposable 3-way stopcock, PC, female × female × male locking (Cole-Parmer, catalog number: UX-48523-36)
11. Dissection tools (see Figure 1 for details)
a. Straight operating scissors: sharp/blunt blades (Figure 1, #5) (Fine Science Tools, catalog number: 14001-12)
b. Student anatomical narrow forceps (Figure 1, #6) (Fine Science Tools, catalog number: 91102-12)
c. Straight dissecting scissors (Figure 1, #7) (World Precision Instruments, catalog number: 14393)
d. Codman Kelly classic forceps, curved, 5-1/2′′ (Figure 1, #8) (Codman, catalog number: 32-4021)
e. Dumont forceps/tweezers pattern #3 (Figure 1, #9) (Stoelting, catalog number: 52100-03)
f. Dissecting scissors (curved) (Figure 1, #10) (Fine Science Tools, catalog number: 14082-09)
g. Double-ended round and tapered micro spoon (Figure 1, #11) (MicroSpatulas.com, catalog number: FD-21-401-10)
12. FalconTM 15 mL conical centrifuge tubes (Fisher Scientific, catalog number: 14-959-53A)
13. GATTA bead nanorulers (GATTAquant, Various distances, https://www.gattaquant.com/)
14. Kimwipes (KimTech, catalog number: 34120)
15. Low-profile microtome blades DB80 LS (Leica, catalog number: 14035843488)
16. Microscope slides: FisherbrandTM SuperfrostTM Plus stain slides (Fisher Scientific, catalog number: 22-034979)
17. Nalgene rapid-flow filters 0.45 μm (Thermo Scientific, catalog number: 166-0045)
18. Peel-A-Way disposable embedding molds 12 mm × 12 mm × 20 mm (Fisher Scientific, catalog number: 12-20)
19. Plastic staining rack (Mopec, catalog number: SP234)
20. Polyurethane ice bucket (Fisher Scientific, catalog number: 02-591-45)
21. Razor blades (Stanley, catalog number: 11-515)
22. Simport Scientific StainTray slide staining system (Simport Scientific, catalog number: 22-045-035)
23. Specimen disc (Leica, catalog number: 14047740044)
24. Sterile Exel International 25 G scalp vein butterfly set (Fisher Scientific, catalog number: 14-840-37)
25. Superfrost Plus microscope slides (Fisher Scientific, catalog number: 12-550-15)
26. Tygon E-3603 (Tygon, catalog number: AC00005)
27. United Scientific 100 mL stainless steel beaker (Fisher Scientific, catalog number: S139215)
28. Vinyl dissecting pad (Carolina, catalog number: 629006)
29. Wheaton Coplin staining jar (DWK Life Sciences, catalog number: UX-48585-20)
30. Zerostat anti-static instrument (Millipore Sigma, catalog number: Z108812)
Equipment
1. Accumet AE150 Benchtop pH Meter (Fisher Scientific, catalog number: 13-636-AE150)
2. Down-draft table for perfusions (fume hood will suffice)
3. Standard Infuse/Withdraw PHD Ultra syringe Pump (Harvard Apparatus, catalog number: 70-3007)
4. Vetflo Traditional Anesthesia System with Vaporizer (Kent Scientific, model: 13-005-201)
5. Ultrasonic Liquid Processors (Misonix, model: XL-2000)
6. Leica CM3050 cryostat (Leica, model: CM3050S, reference number:14047033518)
7. Leica Stellaris 8 Confocal and tau-STED microscope system (Leica Microsystems, Mannheim, Germany)
Software and datasets
1. LAS X (Leica, Version 4.8.2.295667, license required, available on Leica Stellaris 8 instruments)
2. Fiji (NIH, open-source platform for biological image analysis, ImageJ, Version 2.16/1.54P, free to download: https://imagej.net/software/fiji/downloads) [30]
3. Imaris (Oxford Instruments, Version 11.0.0, license required; free version from Imaris Viewer is available at: https://imaris.oxinst.com/imaris-viewer)
Procedure
文章信息
稿件历史记录
提交日期: Dec 23, 2025
接收日期: Feb 24, 2026
在线发布日期: Mar 5, 2026
出版日期: Apr 5, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
如何引用
Scripter, J., Skeens, A., Jones, G., Akter, Y. and Hruska, M. (2026). 3D STED Super-Resolution Imaging Strategy for Visualizing Synaptic Nano-architecture in Brain Cryosections. Bio-protocol 16(7): e5644. DOI: 10.21769/BioProtoc.5644.
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分类
神经科学 > 神经解剖学和神经环路 > 免疫荧光
细胞生物学 > 细胞成像 > 超分辨率成像
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