Whole-mount staining of mouse colorectal cancer organoids and fibroblast-organoid co-cultures

Summary

Imaging organoid culture provides an excellent tool for studying complex diseases such as cancer. However, retaining the morphology of intact organoids for immunolabeling has been challenging. Here, we describe a protocol for immunofluorescence staining in intact colorectal cancer organoids derived from mice. We also describe additional steps for co-culture with mouse fibroblasts to enable the study of interactions with other cellular components of the tissue microenvironment.

For complete details on the use and execution of this protocol, please refer to Martinez-Ordoñez et al. (2023).¹

Graphical abstract

Highlights

• •Steps for appropriate organoid seeding and fixation
• •Protocol for whole-mount staining of 3D organoids grown in semi-suspension and in matrix
• •Analysis of epithelial-stromal interactions by staining of 3D co-cultures

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Imaging organoid culture provides an excellent tool for studying complex diseases such as cancer. However, retaining the morphology of intact organoids for immunolabeling has been challenging. Here, we describe a protocol for immunofluorescence staining in intact colorectal cancer organoids derived from mice. We also describe additional steps for co-culture with mouse fibroblasts to enable the study of interactions with other cellular components of the tissue microenvironment.

Before you begin

This protocol outlines how to fix, permeabilize and perform antibody-based immunofluorescence staining of 3D whole-mount organoids for analysis by confocal microscopy. This protocol allows visualization of organoid proliferation, differentiation, cellular identity, and/or interactions with other cellular components in a co-culture system. This protocol uses colorectal cancer (CRC) mouse tumor organoids (MTOs),² normal small intestinal organoids from Prkci^f/fPrkcz^f/f;Villin-Cre^ERT2,³ and small intestinal fibroblasts isolated from Prkci^f/fPrkcz^f/f (Fib-WT) mice.¹

This protocol builds on (Ineveld et al.).⁴

Institutional permissions

Animal handling and experimental procedures conformed to institutional guidelines and were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee.

Organoid culture

Timing: 4 days

In semi-suspension culture:

• 1.Collect the CRC organoids or normal intestinal organoids in semi-suspension using a 1 mL filtered tip from a 6-well low-attachment plate and transfer it to a 1.5 mL tube.
• 2.Centrifuge on a tabletop microcentrifuge for 6–7 s to pellet the organoids.
• 3.Remove the supernatant. Wash the CRC organoids or normal intestinal organoids once with PBS 1× and centrifuge on a tabletop microcentrifuge for 6–7 s to pellet the organoids.
• 4.
  Disaggregate organoids:

  • a.
    For CRC organoids: add 1 mL of TrypLE per well and incubate for 15 min at 37°C, 800 rpm in an orbital shaker until single-cell suspension.
  • b.
    For normal intestinal organoids: remove the supernatant and add 500 μL organoid culture media with 10 μM of ROCK inhibitor (Y-27632) for each 12-well plate, pipette up and down with a 200 μL filtered tip for mechanical disaggregation.
• 5.For CRC organoids, centrifuge on a tabletop microcentrifuge for 6–7 s to pellet organoids.
• 6.Remove the supernatant and resuspend the CRC single-cell suspension in 1 mL of organoid medium.
• 7.Count the CRC single cells using a hemocytometer or automated cell counter.
• 8.
  Seed organoids:

  • a.
    For CRC organoids: resuspend 2 × 10⁵ single-cell organoids in organoid culture media with 2.5% gel matrix for each 6-well low-attachment plate.
  • b.
    For normal intestinal organoids: cell passage should be 1:4 for each 6-well plate.
• 9.Culture the CRC organoids or the normal intestinal organoids for 4 days changing the media every 48 h.

Note: For growing normal intestinal organoids in semi-suspension, we recommend increasing the gel matrix to 7% to improve growth.

In gel matrix culture:

• 10.Collect the normal intestinal organoids from one 12-well plate by disaggregating the gel matrix with 1 mL ice-cold PBS 1× and transferring the organoids to a 1.5-mL tube.
• 11.Centrifuge on a tabletop microcentrifuge for 6–7 s to pellet the normal intestinal organoids.
• 12.Remove the supernatant. Wash the organoids once with PBS 1× and centrifuge on a tabletop microcentrifuge for 6–7 s to pellet the organoids.
• 13.Remove the supernatant and add 500 μL organoid culture media with 10 μM of ROCK inhibitor (Y-27632) for each 12-well plate, pipette up and down with a 200 μL filtered tip for mechanical disaggregation.
• 14.Centrifuge on a tabletop microcentrifuge for 6–7 s to pellet the normal intestinal organoids.
• 15.Cell passage should be 1:4 for each 12-well plate. Resuspend the normal intestinal organoids in 45 μL of organoid media. Add 105 μL of gel matrix.
• 16.Plate 7 domes of 20 μL for each well from a 12-well plate.
• 17.Add 1 mL of organoid media. Culture the normal intestinal organoids for 4 days changing the media every 48 h.

CRITICAL: For normal organoids, mechanically disaggregating the organoids in organoid culture media with 10 μM of ROCK inhibitor (Y-27632) is recommended instead of enzymatic disaggregation, which might lead to impaired organoid formation.

Note: We recommend culturing CRC organoids in semi-suspension, as they will grow faster, and it will save gel matrix.

Organoid seeding for staining

Timing: 1–2 h

CRC organoids and normal intestinal organoids can either be grown in semi-suspension with a low concentration of gel matrix (2.5%) in low-attachment plates or inside a gel matrix dome (66% gel matrix in organoid culture media). When co-cultured with fibroblasts, organoids are seeded in a gel matrix dome on 8-chamber glass slides to avoid fibroblast loss (Figures 1A and 1B).

Note: Growth in semi-suspension is highly recommended for faster organoid growth and harvesting.

• 18.Before starting, place the desired plate in a 37°C incubator, thaw the gel matrix on ice, and pre-warm TrypLE and organoid culture media in a 37°C water bath for 1 h.
• 19.
  Collect the organoids:

  • a.
    From organoids in semi-suspension: use a 1 mL filtered tip and transfer them into a 1.5 mL tube.
  • b.
    For in-gel matrix organoids: disaggregate the gel matrix with 1 mL ice-cold PBS 1× and transfer the organoids to a 1.5 mL tube.
• 20.Centrifuge on a tabletop microcentrifuge for 6–7 s to pellet the organoids.
• 21.Wash the organoids once with PBS 1× and centrifuge on a tabletop microcentrifuge for 6–7 s to pellet the organoids.
• 22.
  Organoids disaggregation:

  • a.
    For CRC organoids: add 1 mL of TrypLE per well and incubate for 15 min at 37°C, 800 rpm in an orbital shaker until single-cell suspension.
  • b.
    For normal intestine organoids: add 1 mL organoid culture media with 10 μM of ROCK inhibitor (Y-27632), pipette up and down with a 200 μL filtered tip for mechanical disaggregation.

CRITICAL: Disaggregate the normal organoids in organoid culture media with 10 μM of ROCK inhibitor (Y-27632) instead of use enzymatic disaggregation, to avoid impaired organoid formation.

• 23.For CRC organoids: centrifuge on a tabletop microcentrifuge for 6–7 s to pellet the organoids.
• 24.Resuspend the CRC single-cell suspension in organoid culture media.
• 25.Count the cells using a hemocytometer or automated cell counter.
• 26.
  Seeding:

  • a.
    Seed 50–100 cells/μL in 500 μL of organoid culture media, per well, in a 24-well low-attachment plate for monoculture experiments.
  • b.
    Mix 50–100 cells/μL organoids with 5000 fibroblasts in a 50 μL dome (66% gel matrix in media) on an 8-chamber glass slide. After the matrix polymerizes, fill each well with 200 μL of organoid culture media (See Methods video S1).

Note: The growth rate of organoids and fibroblasts will vary for other types of cells, seeding density must be determined before starting the staining procedure. It is recommended to start with at least 500 organoids (See problem 4) (Figure 2A).

CRITICAL: Ensure the gel matrix is always kept on ice and mix it thoroughly before aliquoting and plating.

Figure 1.

Schematics of organoid seeding and culturing

(A) Schematic representation of the organoid culture system in the gel matrix domes in monoculture. An example of MTOs grown in monoculture. Scale bars 200 μm.

(B) Schematic representation of the organoid culture system in the gel matrix domes in co-culture. An example of MTOs grown in co-culture with intestinal fibroblasts after 4 days. Scale bars 200 μm.

(C) Schematic representation of the organoid culture system in semi-suspension. An example of MTOs grown in monoculture in semi-suspension. Scale bars 200 μm.

Figure 2.

Harvesting of MTOs before staining

(A) Images showing MTOs after 4 days of culture in a 24-well plate.

(B) An example of an appropriate size of MTO pellet after centrifugation in a 15 mL tube at 70 × g for 3 min at 4°C.

• 27.Refresh the organoid culture media after 2–3 days, depending on the organoid growth.
• 28.After 4 days, check the organoids under the microscope (Figure 1B). It is recommended not to culture the organoids for more than 4–5 days to prevent overgrowth and organoid collapse (See problem 1).

Alternatives: This protocol has been optimized with Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix. However, several alternatives such as BME (Cultrex) may also be suitable.

Preparation of buffers

Timing: 1 day

We recommend preparing and aliquoting in advance the necessary reagents for gel preparation to facilitate the workflow. All reagents used in this protocol should be molecular biology-grade.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dulbecco’s Phosphate-Buffered Salt Solution 1× (PBS)	Corning	Cat# 21-031-CV
Advanced F12/DMEM	Thermo Fisher Scientific	Cat# 12634020
Glutamax supplement (100×)	Thermo Fisher Scientific	Cat# 35050061
HEPES	Thermo Fisher Scientific	Cat# 12587001
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	Cat# 15140122
Recombinant murine R-Spondin 1	R&D	Cat# 3474-RS-050
Recombinant murine Noggin	PeproTech	Cat# 250-38
Murine EGF	Thermo Fisher Scientific	Cat# PMG8045
B27 supplement (50×)	Thermo Fisher Scientific	Cat# 17504001
Y-27632	Tocris	Cat# 1254
Matrigel™ GFR Membrane Matrix	Corning	Cat# 356230
Cultrex Basement Membrane Extract Type 2, Reduced Growth Factor	R&D Systems	Cat#3533
4% Paraformaldehyde in PBS	Thermo Fisher Scientific	Cat# J61899.AK
Sodium dodecyl sulfate (SDS)	Thermo Fisher Scientific	Cat# BP8200100
Ethylenediamine tetra acetic acid 0.5 M (EDTA)	Life Technologies	Cat# 15575020
Tris	Thermo Fisher Scientific	Cat# 152-10
Fructose	Sigma-Aldrich	Cat# F0127
Urea	Thermo Fisher Scientific	Cat# BP169
Tween-20	Sigma-Aldrich	Cat# P1379-500ML
Triton X-100	Alfa Aesar	Cat# J66624
Gelatin	Sigma-Aldrich	Cat# G1890-100G
Normal goat serum	Vector Laboratories	Cat# S-1000-20
Sodium chloride	Thermo Fisher Scientific	Cat# BP358-10
Glycerol	Sigma-Aldrich	Cat# G9012-100ML
Bovine serum albumin (BSA)	Thermo Fisher Scientific	Cat# BP1600-1
PBS 10×	Corning	Cat# 46-013-CM
TrypLE Express	Gibco	Cat# 12604013
Cell Recovery Solution	Corning	Cat# 354253
Antibodies
Rabbit anti-OLFM4; use at 1:50	Cell Signaling	Cat# 14369, RRID: AB_2798465
Rabbit anti-panCK; use at 1:200	Abcam	Cat# ab9377, RRID: AB_307222
Mouse anti-E-CADHERIN; use at 1:200	BD Biosciences	Cat# 610181, RRID: AB_397580
Rabbit anti-PDGFRa; use at 1:50	Abcam	Cat# ab203491, RRID: AB_2892065
Rat anti-CD44; use at 1:100	Bio-Rad	Cat# MCA4703, RRID: AB_2076194
Biotinylated recombinant HA-binding protein (HABP); use at 1:50	EMD Millipore	Cat# 385911
Alexa Fluor secondary antibodies; use at 1:500	Thermo Fisher Scientific	Cat# A10042, A11004, S11223
DAPI; use at 0.5 mg/mL	Life Technologies	Cat# D1306
Experimental models: Cell lines (∗)
Prkcif/fPrkczf/f;Villin-CreERT2 organoids	Tauriello et al.2	N/A
Mouse tumor organoid (MTO)	Tauriello et al.2	N/A
Prkcif/fPrkczf/f Fibroblast (Fib-WT)	Tauriello et al.2	N/A
Software and algorithms
Zen blue	Zeiss	https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html
Other
Zeiss LSM 710 NLO Confocal Microscope	Zeiss	N/A
EVOS M5000 Imaging System	Thermo Fisher Scientific	N/A
Coverslips 22 × 22 mm thickness 1.5	Glove Scientific, Inc.	1404-15
Coverslips 24 × 40 mm thickness 1.5	Thermo Fisher Scientific	12544C
Sally Hansen Insta-Dri Nail Color 553	Amazon	N/A
6-well tissue culture plates ultra-low attachment	Corning	Cat# 3471
24-well tissue culture plates ultra-low attachment	Corning	Cat# 3473
24-well tissue culture plates	Thermo Fisher Scientific	Cat# 01-549-765
48-well tissue culture plates	Corning	Cat# 3548
8 chamber slide w/cover RS glass	Lab-Tek	Cat# 154534
Single concavity glass slides	Thomas Scientific	Cat# 1216W21

(∗) The passage number of the cell lines used should be < 25.

Materials and equipment

Normal intestinal organoid medium

Reagent	Final concentration	Amount
Advanced F12/DMEM	N/A	94.49 mL
Glutamax supplement (100×)	1%	1 mL
HEPES	1%	1 mL
Penicillin-Streptomycin (10,000 U/mL)	1%	1 mL
B27 supplement (50×)	1%	2 mL
Murine EGF	50 ng/mL	500 μL
Recombinant murine R-Spondin -	1,000 ng/mL	4 μL
Recombinant murine Noggin	100 ng/mL	1 μL
Total	N/A	100 mL

Can be stored at 4°C for 1 month.

Stocks: Recombinant murine R-Spondin-1 25 μg/mL in PBS, Recombinant murine Noggin 10 μg/mL in PBS, murine EGF 100 μg/mL.

CRC organoid medium

Reagent	Final concentration	Amount
Advanced F12/DMEM	N/A	94.5 mL
Glutamax supplement (100×)	1%	1 mL
HEPES	1%	1 mL
Penicillin-Streptomycin (10,000 U/mL)	1%	1 mL
B27 supplement (50×)	1%	2 mL
Murine EGF	50 ng/mL	500 μL
Total	N/A	100 mL

Can be stored at 4°C for 1 month.

Stocks: Murine EGF 100 μg/mL.

SDS 20%

Reagent	Final concentration	Amount
SDS	20%	50 g
dH20	N/A	250 mL
Total	N/A	250 mL

Can be stored at 20°C–22°C for 1 month.

Tris 1 M, pH 8

Reagent	Final concentration	Amount
Tris	1 M	60.55 g
dH20	N/A	500 mL
Total	N/A	500 mL

Note: Dissolve 60.55 g of Tris with 42 mL of concentrated (36%–38%) HCl in 300 mL of dH₂0. Adjust the pH to 8 and fill up to 500 mL.

Can be stored at 20°C–22°C for 1 month.

Organoid Wash Buffer (OWB)

Reagent	Final concentration	Amount
PBS 10×	1×	25 mL
BSA	0.2%	0.5 g
SDS 20%	0.01%	0.125 mL
Triton X-100 10%	0.1%	2.5 mL
dH20	N/A	222.3 mL
Total	N/A	250 mL

Can be stored at 4°C for 1 month.

PBST 1×

Reagent	Final concentration	Amount
PBS 10×	1×	100 mL
Tween-20	0.1%	1 mL
dH20	N/A	899 mL
Total	N/A	1 L

Can be stored at 20°C–22°C for several months.

PBS-BSA 1%

Reagent	Final concentration	Amount
PBS 10×	1×	10 mL
BSA	1%	1 g
dH20	N/A	90 mL
Total	N/A	100 mL

Can be stored at 20°C–22°C for up to 2 weeks.

FUnGI medium⁴

Reagent	Final concentration	Amount
Glycerol	50%	110 mL
Tris 1 M, pH 8	10.6 mM	2.2 mL
EDTA 0.5 M	1.1 mM	440 μL
Fructose	2.5 M	99 g
Urea	2.5 M	33.1 g
dH20	9.4% (v/v)	20 mL
Total	N/A	220 mL

Note: Mix 110 mL of glycerol with 20 mL of dH₂O, 2.2 mL of Tris buffer and 440 μL of EDTA. Add 50 g of fructose and mix at 20°C–22°C in the dark until dissolved. When clear, add 49 g of fructose and mix until dissolved. Then add 33.1 g of urea and mix until dissolved. Aliquot in 1.5 mL tubes.

Can be stored at 4°C in the dark.

Embedded organoids blocking buffer (EOBB)

Reagent	Final concentration	Amount
PBS 10×	1×	10 mL
Triton X-100	0.3%	300 μL
Normal serum	2.5%	2.5 mL
Gelatin	1%	1 g
BSA	1%	1 g
dH20	N/A	87.2 mL
Total	N/A	100 mL

Can be stored at 4°C for up to 2 weeks.

Embedded organoids washing buffer (EOWB)

Reagent	Final concentration	Amount
PBS 10×	1×	25 mL
Triton X-100	0.3%	750 μL
dH20	N/A	224.2 mL
Total	N/A	250 mL

Can be stored at 4°C for up to 2 weeks.

TBS 10×

Reagent	Final concentration	Amount
Tris	200 mM	24 g
NaCl	1.5 M	88 g
dH20	N/A	1 L
Total	N/A	1 L

Adjust pH to 7.6 using HCl. Can be stored at 20°C–22°C for several months.

TBST 1×

Reagent	Final concentration	Amount
TBS 10×	1×	100 mL
Tween-20	0.05%	0.5 mL
dH20	N/A	899.5 mL
Total	N/A	1 L

Can be stored at 20°C–22°C for several months.

Step-by-step method details

Organoid immunofluorescence staining in semi-suspension

Timing: 2 days

In this section, we describe how to harvest organoids in monoculture grown in the gel matrix or in semi-suspension and how to perform fixation, staining, and mounting steps for confocal imaging.

• 1.
  Washing and harvesting of organoids grown in semi-suspension.

  • a.
    Cut the end of a 1 mL tip and pre-coat it with 1% BSA-PBS.
  • b.
    Collect the culture medium containing organoids from a 24-well low-attachment plate in 1.5 mL Eppendorf tubes.
  • c.
    Centrifuge on a tabletop microcentrifuge for 6–7 s.

    CRITICAL: Cutting the end of a 1 mL tip increases its diameter to avoid morphological disruption of organoids.

    Alternatives: If you are starting from a 6-well plate or those with higher volume, centrifugation can be performed in 15 mL tubes at 70 × g for 3 min at 4°C to avoid organoid aggregation.

  • d.
    Discard the supernatant and keep the organoid pellet on ice. Resuspend in 1 mL of ice-cold PBS 1×.
  • e.
    Transfer the organoids to a 15 mL tube precoated with 1% BSA-PBS and add ice-cold PBS 1× up to 10 mL.
• 2.
  Washing and harvesting of organoids grown in gel matrix.

  • a.
    Remove growth organoid culture media from wells of organoids in 24-well plates by vacuum aspiration.
  • b.
    Add 0.5 mL of ice-cold cell recovery solution to each well.
  • c.
    Shake on a rocking shaker, 60 rpm for 60 min at 4°C.
  • d.
    Cut the end of a 1 mL tip and pre-coat it with 1% BSA-PBS.
  • e.
    Resuspend the organoid suspension 10 times using a 1 mL tip.
  • f.
    Transfer the organoids to a 15 mL tube precoated with 1% BSA-PBS and fill up to 10 mL with ice-cold PBS 1×.

Note: The gel matrix should be totally dissociated, and organoids should be clearly floating. If not, the incubation time should be increased (steps b-c).

• 3.
  Fixation and blocking of organoids.

  • a.
    Spin down the tube at 70 × g for 3 min at 4°C. Remove the supernatant without disturbing the pellet (Figure 2B).

    Note: We recommend keeping 1 mL of volume before the complete removal of the supernatant. To prevent loss of organoids, it is recommended to use a 10 μL tip on top of a 1 mL tip.

  • b.
    Gently resuspend the pellet of organoids in 1 mL of 4% PFA, using a 1 mL tip precoated with 1% BSA-PBS.
  • c.
    Incubate on ice for 45 min.

    Note: Halfway through the incubation period, gently resuspend the organoids using a 1 mL pre-coated tip.

  • d.
    Fill the tube with 10 mL of ice-cold PBST 1×. Gently mix by swirling the tube.
  • e.
    Incubate for 10 min at 4°C. Spin down the organoids at 70 × g for 5 min at 4°C. Remove the supernatant.
  • f.
    Resuspend the pellet in 1 mL of cold Organoid Wash Buffer (OWB) and incubate at 4°C for 15 min.

    Pause point: Fixed organoids can be stored at 4°C in OWB for several days, but it is recommended to proceed to the next steps as soon as possible.

• 4.
  Preparation of fixed organoids for staining.

  • a.
    Pre-coat a 48-well plate with 1 mL of 1% BSA-PBS.
  • b.
    Cut the end of a 1 mL tip and pre-coat it with 1% BSA-PBS.

    Note: 1% BSA-PBS solution can be recycled after coating.

  • c.
    Dispense 1/4–1/2 of the preparation of organoids to each well.
  • d.
    Check the number of organoids per well under the microscope. Add more organoids to the well if needed (Methods video S2).

    CRITICAL: The well should contain at least 300–500 organoids for confocal imaging.
  • e.
    Let the organoids sit for 2–3 min at RT. Remove liquid from the top, without removing the organoids. Leave ∼100 μL of volume in the well.

    Note: To estimate how much volume to remove, add 100 μL of buffer into an unused well and compare it to the amount of liquid left (Methods video S2).

• 5.
  Preparation and incubation of primary antibody solution.

  • a.
    Prepare a 2× solution of primary antibody in OWB. Per each sample in a 48-well plate well, prepare 100 μL of 2× antibody solution (for instance, if your working dilution is 1:50, prepare 100 μL of dilution 1:25).

    CRITICAL: Centrifuge the primary antibody briefly before using it to avoid undesired particulates.

  • b.
    Add 100 μL of the antibody solution carefully to the wall of each well.
  • c.
    Incubate the organoids with the primary antibody solution at 4°C on a horizontal rocker for 16 h.
• 6.
  Washing primary antibody.

  • a.
    Fill each well by adding ∼700 μL of OWB to the wall.

    CRITICAL: If the organoids are stuck together, mix them briefly by pipetting with a cut 1 mL tip, pre-coated with 1%BS-PBS.

  • b.
    Wash for 2 h at 20°C–22°C on a horizontal rocker.
  • c.
    Wait for the organoids to settle to the bottom for 2–3 min.
  • d.
    Remove liquid from the top, without removing the organoids, leaving ∼200 μL of volume in the well.
  • e.
    Repeat the wash step (5a) 3 times. The organoids can stay in OWB until adding secondary antibody.

    Note: Add 200 μL of buffer into an unused well to help assess the amount of liquid left.

• 7.
  Preparation and incubation of secondary antibody solution.

  • a.
    Prepare 2× secondary antibody mix with DAPI in OWB. For each sample, prepare 200 μL of 2× antibody. Prepare 2× secondary antibody at dilution 1:250 for a final concentration of 1:500. Prepare 1:2500 for DAPI for a final concentration of 1:5000.
  • b.
    Let the organoids sit for 2–3 min at RT. Remove liquid from the top, without removing the organoids. Leave ∼200 μL of volume in the well.
  • c.
    Add 200 μL of 2× secondary antibody mix carefully to the wall of each well.
  • d.
    Cover the plate in aluminum foil and incubate at 4°C on a horizontal rocker for 16 h.

CRITICAL: Centrifuge the secondary antibody briefly before using it to avoid undesired particulates.

• 8.
  Washing secondary antibody.

  • a.
    Fill each well by adding ∼700 μL of OWB to the wall.
  • b.
    Wash for 2 h at 20°C–22°C on a horizontal rocker.
  • c.
    Wait for the organoids to settle to the bottom for 2–3 min.
  • d.
    Remove liquid from the top, without removing the organoids, leaving ∼200 μL of volume in the well.
  • e.
    Repeat the wash step (7a) 3 times.

CRITICAL: If the organoids are stuck together, mix them briefly by pipetting with a cut 1 mL tip,pre-coated with 1%BS-PBS.

CRITICAL: Keep the organoids protected from light in all steps.

Note: Add 200 μL of buffer into an unused well to help assess the amount of liquid left.

• 9.
  Mounting of the stained organoids on slides.

  • a.
    Clean concave slides with 70% ethanol and label them with tapes.

    Note: Make sure that the side with the concave surface is facing up.

  • b.
    Let the organoids settle for 2–3 min at 20°C–22°C.
  • c.
    Pre-coat 1.5 mL tubes with 1 mL of 1% BSA-PBS.
  • d.
    Transfer the organoids into the pre-coated tubes using a cut 1 mL tip pre-coated with 1%BSA-PBS.
  • e.
    Centrifuge the organoids at 100 × g for 2 min. A small pellet should be observed after the centrifugation.
  • f.
    Accommodate a 10 μL tip in a 1 mL tip and carefully remove as much liquid as possible without disturbing the organoids.
  • g.
    Heat the FUnGI mounting medium at 95°C for ∼1 min until it is slightly warm and liquified.

    Note: Prepare more than one tube of FUnGI mounting medium. It is recommended to prevent cold-warm cycles of the mounting medium when mounting several types of organoids.

  • h.
    Using a cut 200 μL tip, pre-coated with 1%BSA-PBS add 70 μL of FUnGI mounting medium to the organoids and take all the organoids.
  • i.
    Add the organoids to the middle of a clean concave slide, without introducing bubbles.

    Note: If bubbles are introduced in FUnGI mounting medium try to remove them by aspirating with a 200 μL tip without disturbing the organoids.

  • j.
    Carefully place a square coverslip onto the slide, without introducing bubbles.
  • k.
    Carefully seal the edges of the coverslip using nail polish, without moving the coverslip. Make sure to seal the coverslip well.
  • l.
    Let the slides dry at 20°C–22°C for 1 h and proceed to imaging and analysis (Figures 3 and 4).
    See Methods video S3.

    Pause point: The stained slides can be stored at 4°C in dark for up to a week. However, we recommend acquiring the images as soon as possible.

Figure 3.

Whole-mount immunofluorescence staining of whole-mount intestinal normal organoids grown in a gel matrix dome

(A) A representative confocal image of intestinal organoids stained for DAPI (purple), OLFM4 (yellow), and E-cadherin (turquoise). Single Z-stack using a 25× objective. Scale bars 100 μm.

Figure 4.

Whole-mount immunofluorescence staining of CRC organoids grown in semi-suspension

(A) A representative confocal image of CRC organoids stained for DAPI (purple), panCK (yellow), and E-cadherin (turquoise). Scale bars 100 μm.

Organoid immunofluorescence staining in gel matrix

Timing: 2 days

This section describes how to fix, block, incubate with antibodies, and how to mount the slides for confocal imaging for organoids in co-culture inside the gel matrix.

• 10.
  Washing and fixing the organoids and co-cultured cells.

  • a.
    From 8-chamber slide, remove the medium from the wells and wash with 1 mL of PBS 1× without disrupting the gel matrix.
  • b.
    Fix in 500 μL of 4% PFA for 1 h at 20°C–22°C.
  • c.
    Wash 3 times with PBS 1×.

CRITICAL: Do not disrupt the matrix to avoid organoid and cell loss.

Note: After fixation, the gel matrix dome will collapse to the bottom and the cells inside will be attached to the glass surface. It is important to fix for at least 1 h to collapse the matrix dome.

See Methods video S4.

• 11.
  Permeabilization and blocking.

  • a.
    Permeabilize in 500 μL of PBS with 0.3% Triton-X-100 for 15 min at 20°C–22°C.
  • b.
    Block in EOBB for 1 h at 20°C–22°C.

Pause point: Fixed and permeabilized co-cultures can be stored at 4°C in EOBB for several days, but it is recommended to proceed to the next steps as soon as possible.

• 12.
  Incubation with primary antibody.

  • a.
    Remove all the liquid carefully from the corner of each well. The gel matrix is flattened at the bottom of the 8-chamber slide (see Methods video S4).
  • b.
    Prepare 1× primary antibody mix in EOWB. For each well, prepare 125–150 μL of 1× antibody.
  • c.
    Add 125–150 μL of antibody carefully to the wall of each well.
  • d.
    Incubate at 4°C on a horizontal rocker for 16 h.

Note: Usually antibody dilution will range from 1:50 to 1:100 and is usually higher than in the procedure without the gel matrix.

CRITICAL: Centrifuge primary antibodies briefly before using them to avoid particulates.

Note: Using an antibody to specifically stain epithelial cells (e.g., pan-CK or E-cadherin) will help to distinguish organoids from stromal cells.

• 13.
  Washing primary antibody.

  • a.
    Remove all the primary antibody solution from the corner of each well, without touching the bottom of the 8-chamber slide.
  • b.
    Fill each well by adding ∼700 μL of EOWB to the wall.
  • c.
    Wash for 2 h at 20°C–22°C on a horizontal rocker.
  • d.
    Repeat the wash step (4b) 3 times.
• 14.
  Preparation and incubation of secondary antibody solution.

  • a.
    Prepare 1× secondary antibody mix with DAPI in EOWB. For each sample, prepare 125–150 μL of 1× antibody.
  • b.
    Add 125 μL of 1× secondary antibody mix with DAPI carefully to each well.
  • c.
    Cover the plate with aluminum foil and incubate at 4°C on a horizontal rocker for 16 h.

Note: Recommended 1× antibody dilution is 1:500. Recommended DAPI dilution is 1:5000.

Alternatives: Secondary antibody incubation can be performed for 2 h at 20°C–22°C.

• 15.
  Washing secondary antibody.

  • a.
    Remove all the secondary antibody solution from the corner of each well, without touching the bottom of the 8-chamber slide.
  • b.
    Fill each well by adding ∼700 μL of EOWB to the wall.
  • c.
    Wash for 2 h at 20°C–22°C on a horizontal rocker.
  • d.
    Repeat the wash step (6b) 3 times.
• 16.
  Mounting of the stained organoids on slides.

  • a.
    Heat the FUnGI mounting medium at 95°C for ∼1 min until it is slightly warm and liquified.
    Remove the washing buffer and carefully remove the separating walls from the slide (Methods video S5).
  • b.
    Carefully add 100 μL of FUnGI mounting medium onto the slide without introducing bubbles.

    Note: If bubbles are introduced in FUnGI mounting medium try to remove them by aspirating with a 200 μL tip without disturbing the collapsed gel matrix domes.

  • c.
    Carefully place a coverslip onto the slide, without introducing bubbles.
  • d.
    Carefully seal the edges of the coverslip using nail polish, without moving the coverslip. Make sure to seal the coverslip well.
  • e.
    Let the slides dry at 20°C–22°C for 1 h and proceed to imaging and analysis (Figure 5).

    Note: The domes should have collapsed enough to not be squished by the coverslip. The coverslip will contact the collapsed domes.

    Note: Every gel matrix dome will be attached to the bottom of each chamber and will be independently examined under confocal microscopy. See Figure 6.

Figure 5.

Immunofluorescence staining of whole-mount CRC organoids co-cultured with intestinal fibroblasts

(A) A representative confocal image of organoid co-culture stained for DAPI (blue), Hyaluronan (yellow), PDGFRα (cyan), and CD44 (magenta). Scale bars 100 μm.

Figure 6.

Example of confocal parameters for organoid imaging

(A) Zen Black software screenshots of common parameters for Pan-CK (Green), E-Cadherin (Red), and DAPI (Blue).

Expected outcomes

In this protocol, we describe a step-by-step guide for immunolabeling of intact organoids in two different culture settings, without or inside the gel matrix (Figures 3, 4, and 5). These organoids retain the molecular markers and differentiation capacity of epithelial cells in addition to their morphology. Whole-mount staining of organoids is technically challenging and many steps may result in a significant sample loss. Here, we detailed several alternatives to prevent these limitations, providing a versatile and highly reproducible tool to study molecular mechanisms of epithelial homeostasis and malignant transformation and complex cellular interactions between epithelial and stromal cells (Figure 5).

Limitations

Organoid models are very useful tools that successfully recapitulate some of the characteristics of the tissue of origin. However, timing and culture conditions must be optimized for different organoids and cell lines. The organoid semi-suspension system is not suitable for all organoid cultures, especially those composed of normal epithelial cells. For staining inside the gel matrix, tumoral organoid lines and stromal cells such as fibroblasts can grow faster and digest the matrix, disrupting the co-culture system. For both methods, all primary antibodies need to be derived from different species, limiting the number of targets that can be analyzed simultaneously.

Troubleshooting

Problem 1

The organoid structure is broken and/or not well-defined (before you begin step 28).

Potential solution

We recommend performing organoid staining after 4 days of culture to prevent organoid collapse, especially in normal organoids with a more structural differentiation process (e.g., normal intestinal organoids). Avoid excessive pipetting and enzymatic dissociation for normal epithelial organoids.

Because FUnGI can lead to the collapse of the organoids, it is recommended to visualize the organoid staining as soon as the slides are mounted.

Problem 2

Organoids clump together for staining in suspension, making imaging of individual organoids difficult (major steps 2b, 3f, 6a, 8 and 9d).

Potential solution

Ensure that the matrix gel is totally dissociated, and the organoids are freely suspended through all steps (especially in mounting media). Before mounting, gently pipette up and down with a cropped 1 mL tip. Do not add more than 50–100 organoids per slide. Especially when using normal intestinal organoids, make sure to optimize the density of organoids per slide to avoid clumping.

Problem 3

The gel matrix starts to shrink in the co-culture due to the digestion of the gel matrix by fibroblasts, which disrupts organoid growth. The experiment must be terminated early (before you begin, organoid seeding for staining).

Potential solution

Optimize the co-culture system for different cell types before the staining procedure. Common parameters that can be optimized are cell ratio, cell number, and culture time.

Problem 4

There is not enough material for organoid staining in suspension before or after the staining procedure (before you begin, organoid seeding for staining and major steps 1a, 2d, 2f, 3, 8c).

Potential solution

Culture extra organoids in case of loss of material. Coat the wells, tips, and tubes with PBS-BSA 1% before pipetting the organoids. Cut the pipette tip. Ensure that the organoids sink to the bottom of the well before taking out the washes.

Problem 5

Bubbles after mounting interfere with imaging of the organoids (major step 9i).

Potential solution

Try to avoid pipetting bubbles onto the slide. If there are too many bubbles, carefully remove them using a pipette tip without touching the mounting medium. Carefully coverslip without introducing bubbles if possible.

Problem 6

Staining for one or more targets is nonoptimal (before you begin, organoid seeding for staining, major steps 5c, 9a, 11a, and 12d).

Potential solution

Before staining multiple targets simultaneously, we recommend optimizing the staining condition for each target individually. Steps that can be optimized:

• •Organoid size: overgrown organoids will collapse and lose morphology. Optimize organoid size before staining experiments. We have performed successful immunolabeling of intact organoids with a size range from 150–250 μm in diameter for normal organoids, and from 200–500 μm in diameter for CRC organoids.
• •Permeabilization: Increasing permeabilization time could improve antibody penetration.
• •Antibody incubation: Test different concentrations of the target antibodies as well as incubation time to improve the staining.
• •It is highly recommended to use single concavity glass slides rather than regular slides to reduce the possibility of organoid morphology disruption.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Maria T. Diaz-Meco (mtd4001@med.cornell.edu).

Materials availability

This protocol did not generate new unique reagents.

Data and code availability

This study did not generate new datasets.