Histological Processing of Organoids for Immunostaining

Materials and reagents

Biological materials

1. Organoids (300 μm to 3 mm in size) embedded in matrix domes

Reagents

1. Double-deionized water produced by the Synergy water purification system and ion exchange resin water system (Merck Millipore, catalog number: SYNSVHFFR-KIT and Veolia water system, catalog number: CFCFGU201181)

2. Methanol-stabilized formaldehyde 30% in solution (Carl ROTH, catalog number: 4235)

3. Sodium azide (NaN₃) (Sigma-Aldrich, catalog number: S8032)

4. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405)

5. Potassium dihydrogen phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P0662)

6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)

7. Sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O) (Sigma-Aldrich, catalog number: S9390)

8. Glycine, aminoacetic acid (Sigma-Aldrich, catalog number: G8898)

9. Butanol-2-Methyl-1-butanol (Sigma-Aldrich, catalog number: 133051-100ML)

10. Xylene histological grade [C₆H₄(CH₃)₂] (Sigma-Aldrich, catalog number: 534056-500ML)

11. Absolute ethanol (CH₃CH₂OH) (Sigma-Aldrich, catalog number: 1070172511); when necessary, dilute ethanol with double-deionized water

12. Epredia HistoGel specimen processing gel (Epredia, catalog number: HG4000012)

13. Epredia Paraffin Type 9 (Epredia, catalog number: 8337)

14. Hematoxylin GILL II (Diapath, catalog number: CP813)

15. Trichloroacetic acid (Thermo Fisher Scientific, catalog number: 7402)

16. Lithium carbonate (Microm Microtech, catalog number: 45006 29)

17. Eosin (Diapath, catalog number: C0362)

18. Saffron (VWR, catalog number: 27481.105)

19. Diamount mounting medium (Diapath, catalog number: 030400)

20. Dako Target retrieval solution, citrate pH 6.1, 10× (Agilent, catalog number: S1699)

21. ReadyProbes hydrophobic barrier pap pen (Life Technologies, catalog number: R3777)

22. ProLong Gold anti-attenuation mounting medium (Life Technologies, catalog number: P36930)

23. Dako protein block solution, ready to use (Agilent, catalog number: XO909)

24. Dako antibody diluent, ready to use (Agilent, catalog number: SO809)

25. Antibody (depending on application)

Solutions

1. PBS 10× (see Recipes)

2. PBS/glycine (see Recipes)

3. 2% formaldehyde (see Recipes)

Recipes

1. PBS 10×

Dilute 10× solution with double-deionized water for 1× utilization. Store at 4 °C for up to one year.

2. PBS/glycine

Dilute 10× solution with double-deionized water for 1× utilization. Store at 4 °C for up to six months.

3. 2% Formaldehyde

Prepare fresh before use.

Laboratory supplies

1. Complete staining sets: well, cover, slide rack (Epredia, catalog number: 121)

2. Slides for microscopy Epredia SuperFrost Plus Gold (Epredia, catalog number: K5800AMNZ72)

3. Lid of 48-wells plate (Corning, catalog number: 3548)

4. Immunostaining slide moisture chambers (Evergreen, catalog number: 240-9020-Z10)

5. Dissection Fine-Pointed Forceps (Fisherbrand, catalog number: 08-880)

6. 660 mL glass jar (Dutscher, catalog number: PI82PC660)

7. White Pasteurizable lid, TO 82 ring (Dutscher, catalog number: CPAST82BL)

8. Razor blade compatible with microtome (ThermoFisher Scientific, catalog number: 3050835)

9. 12-piece blue HB pencil set (Lyreco, catalog number: 013LYRK7720/1)

10. Epredia Cryotom Cryostat Accessories, Camel HairBrush (Epredia, catalog number: 1910)

11. Epredia Shandon disposable embedding molds 15 × 15 mm (Epredia, catalog number: 41741)

12. Epredia Shandon disposable embedding molds 24 × 30 mm (Epredia, catalog number: 41743)

13. Fisherbrand graduated transfer pipettes, non-sterile, LDPE, clear (Fisher Scientific, catalog number: 13439108)

14. Epredia Cassette II slotted tissue cassettes (Epredia, catalog number: B851729)

15. Biopsy foam pads (Simport Scientific, catalog number: M476-1)

Equipment

1. 115 V Programmable Dry Bath Incubator (Boekel Scientific, catalog number: 115001)

2. Paraffin dispenser (Bio-Optica Milano, catalog number: 40-200-101)

3. Cooling plate (Micro Microtech, catalog number: EC 350-2)

4. Decloaking Chamber^TM NxGen (Biocare Medical, catalog number: DC2012-220V)

5. Block Module, 16 mm tubes (Boekel Scientific, catalog number: 110016)

6. Stuart Mini Gyro-Rocker SSM3 (Fisher Scientific, catalog number: 10034264)

7. Microtome Microm (Epredia, catalog number: HM355S)

8. Epredia^TM STP 120 Spin Tissue Processor (Epredia, catalog number: STP 120)

9. Automated slide stainer Gemini AS (Epredia, catalog number: A81500001)

10. Epifluorescence microscopes (Zeiss, Axio Observer 7 with LED Colibri 7 and HAMAMATSU C11440 digital camera; Nikon, Eclipse Ti2 with DS-Fi3 brightfield camera and DS-Qi2 fluorescence camera)

Procedure

A. Sample preparation

1. Sample fixation

a. Remove culture media and add 2% formaldehyde solution (see Recipe 3) to fix the organoids directly in matrix domes.

b. Incubate for 90 min at room temperature under slow rocking agitation (between 20 and 25 rpm).

Note: A 90-min incubation ensures that formaldehyde efficiently penetrates organoids ranging from 500 μm to 2 mm, ensuring uniform fixation. The fixation time can be adjusted depending on organoid size.

c. Remove 2% formaldehyde and wash three times for 10 min with PBS/glycine (see Recipe 2).

d. Store at 4 °C in PBS with azide 0.1% to prevent microbiological contamination.

Note: PBS with 0.1% azide can be prepared in advance and stored at room temperature for 6 months. Sodium azide is highly toxic and requires the use of proper personal protective equipment. Sodium azide needs to be disposed of in a proper container according to country regulations.

Pause point: Fixed organoids can be stored at 4 °C for 2 months (or 12 months if you add PBS with 0.1% azide every 2 months to prevent drying out the fixed samples).

Note: We recommend including organoids in paraffin soon after fixation to avoid tissue degradation over time.

2. Preparation of fixed samples before inclusion

a. Heat the HistoGel for 30 min at 90 °C with a block module.

b. Make 100 and 250 µL drops of warm liquid HistoGel using transfer pipettes on a 48-well plate lid (Figure 1; Video 1).

c. Gently detach the matrix domes containing the organoids with a metal clip and place them in the HistoGel drop.

Notes:

1. Alternatively, put the organoids directly in the HistoGel drops if you culture your 3D organoids without Matrigel, matrix, or polymer.

2. It is possible to put 2 or 3 matrix domes (each with several organoids) inside one HistoGel drop. This is recommended when the organoid yield per dome is low, e.g., 10 organoids per dome.

d. Wait for 5 min for the HistoGel to cool, then place another droplet of HistoGel on top of each organoid dome.

e. Wait for 30 min for the HistoGel to polymerize and then remove any excess product using the flat side of microdissection forceps or a scalpel.

f. Place the dome containing organoids on a cassette that has been previously covered with biopsy foam.

Note: Adjust the number of domes loaded into each cassette according to the total number of organoids available and the desired representation in the final tissue sections. When organoid yield per matrix dome is low, multiple domes may be embedded within the same cassette to increase the likelihood of interesting organoids during microtomy (up to 4 domes per cassette, or up to 8 depending on mold dimensions). Under standard conditions, 1–2 domes are typically placed in each histology cassette.

g. Close and submerge the cassettes in 70% ethanol overnight at 4 °C.

B. Sample inclusion

1. Dehydration of samples

a. Perform the dehydration of organoids using the Spin Tissue Processor with the following program:

Ethanol 80% 10 min

Ethanol 95% 10 min

Ethanol 95% 10 min

Ethanol 95% 10 min

Ethanol 100% 10 min

Ethanol 100% 10 min

Ethanol 100% 10 min

Butanol 15 min

Butanol 15 min

Butanol 15 min

Paraffin wax 40 min

Paraffin wax 40 min

Notes:

1. Butanol is used here because it allows a gentler dehydration than ethanol or methanol. It reduces tissue shrinkage, which is recommended for fragile tissues such as organoids.

2. The last two paraffin cycles ensure complete and deep penetration of paraffin into Matrigel and organoids. This decreases the risk of ribbon breakage during section processing.

3. Switch on the paraffin dispenser to prepare for the next step and have the melted paraffin ready by the end of the inclusion cycle. Also, turn on the cooling plate approximately 30 min before the next step.

2. Inclusion

a. Transfer the cassettes into the paraffin fountain.

b. Pour liquid paraffin wax into the bottom of a disposable embedding mold.

c. Wait a few seconds above the cooling station for the paraffin to harden.

d. Remove the cassette from the paraffin fountain and transfer the dehydrated HistoGel drops to the base mold.

Note: Incorporate 2–3 HistoGel droplets into the same paraffin block to increase the number of organoids per slide.

e. Place paraffin wax in the mold to fill it.

f. Remove the cassette lid and place the bottom part on top of the mold to create a tissue block.

g. Let the paraffin harden on the cooling station for 15–30 min.

h. Remove the disposable base mold from the paraffin tissue block.

Pause point: Paraffin blocks can be stored at room temperature until sectioning.

Figure 1. Setup of HistoGel inclusion. (A) A first droplet of HistoGel is deposited on the cover of a 24-well plate. (B) Organoids in Matrigel domes are delicately deposited on top of the HistoGel droplet. (C) Representative image of organoids included in HistoGel (1), inside a histological cassette (2), and covered with foam (3).

C. Sample block section

1. Turn on the microtome, position the paraffin block, and secure it with the screws.

2. Bring the paraffin block near the razor blade and lower the blade protection.

Note: The razor blade should be changed at the end of each sectioning session or when the quality of the sliced paraffin ribbon decreases.

3. Align the paraffin block parallel to the razor blade.

4. Select cutting speed and thickness and then cut the tissue into strips.

Notes:

1. We recommend first trying a speed between 10 and 15 mm/s, with a thickness of 5 μm.

2. If the sections crumble, rehydrate the blocks by soaking them in a PBS bath on ice for at least 30 min.

5. Use a paintbrush to gather the paraffin sections and gently float them in a bath of distilled water warmed to 35 °C ± 5 °C.

Note: If the bath temperature exceeds 45 °C, the paraffin might melt again.

6. Place histological sections on a slide and allow them to dry for a few minutes at room temperature.

Note: Slides can be kept in a slide box at room temperature until they are needed again.

D. Sample immunostaining

D1. Dewaxing

1. Incubate the slide for 1 h at 60 °C in a dry oven and proceed to dewax by immersing it in two successive 15-min xylene baths.

2. Rehydrate slides by immersing them sequentially in the following series of baths:

100% ethanol for 5 min

95% ethanol for 5 min

85% ethanol for 5 min

70% ethanol for 5 min

Deionized water 2 times for 2 min

Note: Once the procedure is complete, do not leave the slides in deionized water or let them dry out, as this can cause the tissue to degrade. Proceed immediately to slide coloration or immunofluorescence staining.

D2. Hematoxylin and eosin coloration

1. H&E staining can be performed using an automated slide stainer. The staining procedure can then be carried out as follows:

Hematoxylin 3 min

Deionized water 3 min

Trichloroacetic acid 45 s

Deionized water 1 min

Lithium carbonate 20 s

Deionized water 1 min

Eosin 6 min

Deionized water 1 min

Ethanol 100% 2 baths

Saffron 2 min

2. Proceed to slide dehydration by bathing them in the following series of baths:

70% ethanol for 5 min

85% ethanol for 5 min

100% ethanol for 5 min

Xylene for 5 min

Xylene for 5 min

Mount slides with Diamount mounting medium or other non-aqueous mounting medium.

D3. Immunofluorescence staining

1. Antigen unmasking

Critical: Paraffin-embedded tissues typically require an antigen unmasking step before staining to ensure optimal epitope–antibody binding. Various antigen retrieval techniques and solutions are available and should be selected based on the target antigen and the antibody used. The following steps outline the antigen unmasking procedure used to achieve the stainings shown in Figure 2.

a. Dilute 20 mL of Dako Target retrieval solution pH 6 into 180 mL of distilled water.

Caution: Use the Dako retrieval under a hood suitable for CMR substances.

Note: Dako retrieval solution also exists at pH 9. Make a choice based on the antibodies used and their data sheet.

b. Submerge the slide into the Dako retrieval solution and perform heat-induced epitope retrieval in a decloaking chamber at 110 °C for 90 s.

Note: Pressure parameters cannot be set for the decloaking chamber, but this device reaches 25 psi at 110 °C.

c. Discard the Dako Target retrieval solution once it has cooled. Then, rinse the slides with three consecutive PBS baths.

Figure 2. Histology and immunostaining of organoids. (A) Hematoxylin and eosin (H&E) staining (left) and immunofluorescence staining (right) of paraffin-embedded human intestinal organoid sections. Human intestinal organoids are characterized by expression of the hindgut marker CDX2, the epithelial marker Cadherin-1 (CDH1), and the mesenchymal marker Vimentin (VIM). Scale bars: 100 μm. Representative images of >20 biological replicates. (B) H&E staining (left) and immunofluorescence staining (right) of paraffin-embedded human cardioid sections. Left ventricle (LV), right ventricle (RV), and right ventricular outflow tract (RVOT) are stained for the cardiomyocyte marker Troponin T and the ventricular myocytes IRX4. Scale bars: 100 μm. Representative images of >10 biological replicates.

2. Antibody staining

a. Outline tissue sections using a hydrophobic pap pen to help contain reagents.

b. Incubate slides with protein block solution for 1 h in a humidity chamber at room temperature.

c. Aspirate blocking solution.

d. Incubate slides with primary antibodies diluted in Dako protein block solution at 4 °C overnight in a slide moisture chamber.

Note: Follow the antibody datasheet to achieve the most appropriate dilution for the tissue analyzed. Typically, for organoid staining, the dilution range is 1:200–1:1,000, depending on the manufacturer’s instructions.

e. Wash slides in PBS for 10 min with gentle agitation. Repeat the procedure three times.

Note: We advise against washing slides in racks to avoid antibody cross-contamination and to prevent tissue damage or loss during the wash.

f. Incubate slides with secondary antibodies diluted in Dako protein block solution for 1 h at room temperature in a slide moisture chamber.

Note: Follow the antibody datasheet to achieve the most appropriate dilution for the tissue analyzed.

g. Wash the slides in PBS for 10 min, protecting them from light. Repeat this process three times.

h. Incubate slides with 1 μg/mL of DAPI in PBS for 5 min while protected from light.

i. Wash microscope slides in PBS for 3 × 10 min each, while protected from light.

j. Mount microscope slides with coverslips using Prolong Gold mounting medium or another aqueous mounting medium.

Critical: Remove any bubbles that have a refractive index different from that of the mounting medium.

k. Proceed with imaging under an epifluorescence microscope.

Validation of protocol

This protocol has been used and validated in the following research articles:

1. Vales S, Bonneau L, Mahe MM. Differentiating Enteric Glial Cells Within Intestinal Organoids Derived from Human Pluripotent Stem Cells. Methods Mol Biol. (Figure 3)

2. Bonneau L et al. Generation of intestinal and colonic organoids derived from human pluripotent stem cells. Biology of the Cell. (Figures 2b and 3a)

General notes and troubleshooting

Troubleshooting

Problem 1: HistoGel drops shrank after dehydration.

Possible cause: The HistoGel has expired or undergone too many liquefaction cycles (after three liquefaction cycles, the quality of HistoGel cannot be guaranteed).

Solution: Use a fresh tube of HistoGel or a new batch.

Problem 2: Paraffin sections break apart and disintegrate during sectioning.

Possible causes: The room is too hot, the paraffin block is too dry, or the embedding has failed.

Solution: Before sectioning, rehydrate the block by immersing it in PBS at 4 °C for 30 min, or melt the block and re-embed the samples (section B).

Problem 3: Samples peel off the slides after dewaxing by rehydration.

Possible cause: The section is of poor quality, or the blades used are not recommended for this type of experiment.

Solution: Perform new tissue sections and ensure the use of unused, certified blades.

Problem 4: Antigen retrieval is uneven.

Possible cause: The parameters of the decloaking chambers do not match the antibodies used.

Solutions: Adjust the temperature, time, and pressure of the decloaking chamber. Use a Dako Target retrieval solution at a different pH that better suits the antibody, following the manufacturer’s instructions.

Problem 5: Immunofluorescence signal is weak and/or has excessive background fluorescence.

Possible causes: The antibody is too diluted, not specific, or has expired.

Solutions: Try to increase the concentration of the antibody (1:200 or 1:100); alternatively, use a new batch.

Problem 6: During the tissue sectioning process, too many slices are empty, lacking biological material.

Possible cause: The Matrigel droplet did not contain enough organoids.

Solution: Increase the number of HistoGel droplets included in the same histological cassette. If matrix domes contain very few organoids, include up to 4 droplets per histological cassette. This increases the chance of cutting through biological material during sectioning.

Acknowledgments

Conceptualization, N.G., M.M.M., S.V.; Investigation, L.B., V.P., S.V., L.B., A.B., T.N., S.G., L.B., A.K., G.L.; Writing—Original Draft, L.B., V.P., S.V.; Writing—Review and Editing, M.M.M., L.B., V.P., A.G., N.G.; Funding acquisition, M.M.M.; Supervision, S.V., M.M.M.

This work was supported by funding from ANR-17-CE14-0021 (SyNEDI to M.M.M.), ANR-21-CE14-0017 (NeuroPIMM to M.M.M.), ANR-22-CE17-0051 (to N.G., G.L., and S.G.), Fédération Française de Cardiologie (FIBRIL R21173NN to N.G., G.L., and S.G.), a grant from the Medical Research Foundation (FRM - REP202110014143 to M.M.M.), and by the program "Trajectoire Nationale" from Région Pays de La Loire.

We acknowledge Stéphanie Blandin from the MicroPICell core facility (SFR Bonamy, Bi°Core, Inserm UMS 016, CNRS UAR 3556, Nantes, France), member of the Scientific Interest Group (GIS) Biogenouest, IBISA, and the national infrastructure France-Bioimaging supported by the French national research agency (ANR-24-INBS-0005 FBI BIOGEN).

We acknowledge Anne Gaignerie from the ION infrastructure (SFR Bonamy, Bi°Core, Inserm UMS 016, CNRS UAR 3556, Nantes, France), supported by the Infrastructures de Recherche Ligériennes 2022 program from Région des Pays de La Loire.

Competing interests

The authors declare no conflicts of interest.

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