Visualizing Cell Type–Specific Glycan Profiles in Tissue Sections With Glycan Painting

Materials and reagents

Biological materials

1. Formalin-fixed, paraffin-embedded mouse embryo (E10.5) tissue sections (Genostaff, catalog number: HMPS-02)

2. Concanavalin A, CFR405S conjugate (Fujifilm, catalog number: 559-54431)

3. Wheat germ agglutinin, CFR488A conjugate (Fujifilm, catalog number: 557-54231)

4. Tomato lectin (LEL), Dylight594 conjugate (Thermo Fisher, catalog number: L32471)

Note: Store all fluorescent lectin conjugates at 4 °C protected from light. Avoid repeated freeze/thaw cycles. In our laboratory, the conjugates retained staining performance for at least six months under these conditions.

Reagents

1. PathoClean (Fujifilm, catalog number: 161-28321)

2. Ethanol (Fujifilm, catalog number: 0596-00486)

3. 20× Tris-buffered saline (TBS), pH 7.4 (Fujifilm, catalog number: 317-90371)

4. D-PBS(+) preparation reagent (Ca, Mg solution) (100×) (Nacalai Tesque, catalog number: 02492-94)

5. 10× carbo-free blocking solution (Vector, catalog number: SP-5040-125)

6. 10% formalin-neutral buffer solution (Fujifilm, catalog number: 062-01661)

7. 1,4-diazabicyclo[2.2.2]octane (DABCO) (Fujifilm, catalog number: 049-25712)

8. Glycerol (Fujifilm, catalog number: 075-00616)

9. Tris(hydroxymethyl)aminomethane (Fujifilm, catalog number: 207-06275)

10. Kaneyonol (Kaneyo soap laundry glue, Kaneyo); if unavailable, 8% polyvinyl alcohol (PVA) (Sigma, catalog number: P8316) or a commercially available mounting medium can be used as an alternative

Solutions

1. Wash buffer (see Recipes)

2. Blocking buffer (see Recipes)

3. Staining buffer (see Recipes)

4. Mounting medium (see Recipes)

Recipes

Note: All solutions, except for the mounting medium, are freshly prepared before each experiment and should not be reused.

1. Wash buffer (100 mL)

2. Blocking buffer (10 mL)

3. Staining buffer (1 mL)

4. Mounting medium (40 mL)

Note: Kaneyonol or 8% PVA is used as a viscosity-enhancing agent in the mounting medium to maintain an appropriate layer of mounting medium beneath the coverslip. Upon drying, the mounting medium solidifies and helps stabilize the coverslip. The mounting medium is stored in the dark and reused for subsequent experiments.

Laboratory supplies

1. 1.5 mL microtube (FastGene, catalog number: FG-MCT15A)

2. 50 mL disposable centrifuge tube (Iwaki, catalog number: 2342-050N)

3. 100 mL disposable centrifuge tube (Iwaki, catalog number: 2355-100N)

4. Coverslip (Matsunami, catalog number: C022221)

5. A-PAP pen (Matsunami, catalog number: GMP0020)

Equipment

1. Confocal microscopy (AX, Nikon, Tokyo, Japan, model: Nikon AX with NSPARC Super-Resolution Confocal Laser Microscope System)

2. Aspirator (IWAKI, AGC Techno Glass, Shizuoka, Japan, catalog number: VPUMP-140)

Software and datasets

1. Fiji/ImageJ (version 2.16.0, National Institutes of Health (NIH), USA; used for fluorescence image processing, channel merging, RGB analysis, and image quantification

2. NIS-Elements AR (version 6.20, Nikon, Tokyo, Japan); used for image acquisition and processing of confocal microscopy data

Procedure

A schematic overview of the glycan painting protocol from FFPE tissue section preparation to fluorescence image acquisition is shown in the Graphical overview.

A. Deparaffinization of FFPE tissue sections

1. Deparaffinize tissue sections in PathoClean in 50 mL disposable centrifuge tubes, as follows:

a. Immerse sections in PathoClean for 5 min.

b. Transfer sections to fresh PathoClean for 3 min.

c. Transfer sections to fresh PathoClean for 2 min.

d. Transfer sections to fresh PathoClean for 1 min.

2. Rehydrate sections through a graded ethanol series in 50 mL disposable centrifuge tubes, as follows:

a. Immerse sections in 100% ethanol for 5 min.

b. Transfer sections to fresh 100% ethanol for 3 min.

c. Immerse sections in 95% ethanol for 2 min.

3. Rinse slides in double-distilled water and immerse them in TBS buffer in 50 mL disposable centrifuge tubes. The rehydration procedure is performed according to the manufacturer’s recommended protocol for PathoClean (Fujifilm).

B. Blocking

1. To prevent tissue sections from drying, gently wipe excess moisture from the glass slide using a laboratory wipe and draw a hydrophobic barrier around the section with an A-PAP pen. Allow the barrier to dry completely for approximately 1 min at room temperature.

2. After the A-PAP pen barrier has dried, add the blocking buffer to the inner area enclosed by the hydrophobic barrier and incubate for 45 min at room temperature.

C. Lectin staining

1. Wash the blocked slides three times with washing buffer. During each wash, add washing buffer to tissue sections using a micropipette and carefully remove the buffer with an aspirator.

2. Add 400 μL of staining buffer per 2 cm² of the area enclosed by the hydrophobic barrier pen, ensuring that the tissue section is completely covered. Incubate for 60 min at room temperature in the dark or overnight at 4 °C. To prevent evaporation of the staining buffer during incubation, place the slides in a 10-cm Petri dish or a humidified chamber.

3. Wash the slides three times with approximately 400 μL of washing buffer per 2 cm² of tissue area.

4. Immediately after washing, fix the stained sections with 10% formalin-neutral buffer solution for 10 min at room temperature in the dark. This post-fixation is performed to stabilize lectin–glycan interactions and preserve the fluorescent staining patterns during subsequent processing.

5. Wash slides three times with washing buffer under the same conditions as described above.

Caution: 10% formalin-neutral buffer solution is toxic and should be handled in a certified chemical fume hood while wearing appropriate personal protective equipment (gloves, lab coat, and eye protection).

Pause point: If necessary, the incubation can be extended overnight at 4 °C in the dark without affecting staining performance.

Critical: Do not allow the tissue sections to dry at any stage of the procedure. Drying may increase nonspecific background staining and reduce staining reproducibility.

Critical: ConA requires divalent cation for glycan binding. Therefore, the staining buffer should contain Ca²⁺ to ensure optimal staining.

D. Mounting

1. Apply an appropriate amount of mounting medium onto the tissue section and carefully place a coverslip over the sections while avoiding the introduction of air bubbles.

2. Allow the slide to incubate for 3 h at room temperature in a dry and dark place.

Pause point: Mounted slides may be stored at 4 °C, protected from light, for several days before imaging.

E. Image acquisition

For conventional fluorescence microscopy, acquire images using DAPI, GFP, and Texas Red filter sets corresponding to ConA-CFR405S, WGA-CFR488A, and LEL-DyLight 594, respectively. In the case of confocal microscopy, acquire images using the 405, 488, and 561 nm laser lines. Figure 1 shows a composite tiled image generated from 72 confocal images acquired for each fluorescent lectin, together with the corresponding merged image. A 20× objective lens was used to provide a sufficient field of view while maintaining adequate spatial resolution for visualizing and comparing glycan distribution patterns across different tissues and cell populations. Images were acquired at a resolution of 1,024 × 1,024 pixels with two-line averaging and a pixel dwell time of 3.2 μs. The laser power was set to 5.7%, 1.8%, and 10.2% for the 405, 488, and 561 nm lasers, respectively. Detector gain was set to 85, and the pinhole was set to 1.4 Airy units. These settings were kept constant for all images acquired within each experiment. However, laser power, detector gain, and other acquisition parameters were adjusted as needed between samples to obtain optimal image quality while avoiding signal saturation.

Figure 1. Confocal images of a mouse embryo E10.5 section stained using glycan painting. The mouse embryo section was stained with (A) ConA-CFR405S, (B) WGA-CFR488A, and (C) LEL-DyLight594. (D) Merged image of triple staining. (E) H&E-stained image and (F) autofluorescence images of serial sections prepared from the same FFPE block. Scale bar: 1 mm.

Data analysis

Merged images were adjusted for brightness and contrast using FIJI (ImageJ). In this protocol, image analysis was primarily qualitative, focusing on comparisons of glycan-dependent color patterns among different cell populations. The representative images in Figure 1 were selected from an FFPE mouse embryo section, because it contains multiple organs and diverse cell populations within a single section, allowing visualization and comparison of characteristic glycan distribution patterns across different tissues and cell types.

Potential spectral overlap and channel cross-talk should be considered when interpreting fluorescence signals. Therefore, glycan painting is intended for qualitative interpretation of relative RGB color patterns, rather than quantitative comparison of individual fluorophore intensities or absolute fluorescence levels.

Quantitative comparison of fluorescence intensities across different experiments or imaging systems requires standardized reference samples for signal normalization. Because such a standardization system is currently under development, this protocol is intended for the qualitative interpretation of characteristic RGB color patterns rather than the quantitative assessment of absolute fluorescence intensities.

Validation of protocol

This staining protocol was independently repeated in three separate experiments on different days using FFPE mouse embryo sections, yielding reproducible staining results. Representative images from each experiment are shown in Figure 2. The broader applicability of glycan painting has been demonstrated in our previous bioRxiv preprint (Figure 2 from [6]), which described its application to mouse tissue sections at different developmental stages.

Figure 2. Representative images from three independent staining experiments performed on different days. All images were acquired under identical imaging conditions, and similar fluorescence patterns were reproducibly observed across the three experiments. Scale bar: 1 mm.

General notes and troubleshooting

General notes

1. Tissue sections should not be allowed to dry during staining steps.

2. ConA and certain other lectins require divalent cations, such as Ca²⁺, for optimal glycan binding.

3. Commercial mounting reagents compatible with fluorescence imaging, including PVA-DABCO-based formulations, may be used as alternatives to the mounting medium described above.

4. For imaging of large tissue areas, near-infrared nuclear staining DRAQ5 is recommended for reliable focusing.

5. Carbo-free blocking solution is recommended for blocking before lectin staining to avoid an increase in background fluorescence.

Troubleshooting

Problem 1: High background fluorescence.

Possible cause: The tissue section has dried during the staining procedure.

Solution: Avoid drying of sections and perform incubation in a humidified chamber.

Problem 2: Rapid fading of fluorescence.

Possible cause: Excessive exposure to excitation light.

Solution: Minimize light exposure and use an antifade mounting medium when possible.

Problem 3: Uneven staining.

Possible cause: Insufficient reagent coverage on the tissue section.

Solution: Ensure the entire tissue area is completely covered with solution.

Acknowledgments

This research was supported by Management Expenses Grants from AIST, AMED (grant numbers JP23ym0126803 and JP25ak0101254), and the Japan Society for the Promotion of Science (JSPS) under KAKENHI Grants 24K22414 and 25H01226. The author thanks Dr. Kazumi Hirano and Dr. Chiaki Okatani for valuable and helpful advice.

ChatGPT (OpenAI) and Microsoft Copilot (Microsoft) were used for English language editing and proofreading. The authors reviewed all AI-assisted text and take full responsibility for the accuracy and content of the manuscript.

Author contributions

Conceptualization, A.N.; Investigation, A.N.; Writing—Original Draft, A.N.; Writing—Review & Editing, Y.S.; Figure Preparation, A.N. and Y.S.; Funding Acquisition, A.N. and Y.S.

Competing interests

The author declares no competing interests.

Ethical considerations

Commercially prepared tissue sections used in this study were purchased from certified suppliers and were collected and distributed in accordance with the applicable ethical and regulatory standards. Glycan painting uses standard lectins and fluorescent reagents and does not involve genetic manipulation or infectious materials, nor are any of the procedures associated with major biosafety concerns.

References

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