Protocol for Immunofluorescence Staining of Organoids Embedded in Cultrex™ Extracellular Matrices

Set-up of Organoid Basement Membrane Extract Domes

To optimize culture vessel space and make organoid manipulation easier, we recommend culturing organoids embedded in Cultrex BME as domes arranged in wells of a 6-well plate; this may be scaled for larger area plates if needed. Each dome should contain 50 μL of resuspended organoids in BME. In Figure 1, the placement of one dome in the center of a well of a 24-well plate or six domes in each well of a 6-well plate is shown. Once finished, the plate is placed in a tissue culture incubator at 37 °C to polymerize the BME. After the BME has polymerized, 3 mL of organoid culture medium is added to each well. NOTE: The Cultrex BME domes should not touch the sides of the wells.

Organoid Harvesting and Passaging

1. Discard the culture or differentiation medium and wash the well(s) with 5 mL of cold (2-8 °C) PBS. Incubate the organoids with 5 mL of cold (4 °C) Cultrex Organoid Harvesting Solution (R&D Systems, Catalog # 3700-100-01) for 30 to 60 minutes. NOTE: During this time, the plates should be placed inside a container with ice or in a cold room with gentle shaking in order to achieve maximum depolymerization of the Cultrex BME.

2. Once the matrix is dissolved and the organoids are released, transfer the solution to a conical tube and mechanically disrupt the organoids by passing them through a 20 gauge needle attached to a syringe.

3. Centrifuge the fragmented organoids at 500 x g for 5 minutes at 2-8 °C. Discard the supernatant.

4. Wash the cell pellet with 5-10 mL of cold (2-8 °C) 1X PBS, centrifuge again at 500 x g for 5 minutes at 2- 8 °C. Discard the supernatant.

5. After a final centrifugation, discard the remaining PBS and resuspend the organoids in Cultrex BME for re-plating.

Organoid Fixing, OCT Embedding, and Cryosectioning

1. Remove the organoid culture media and wash each well of a 6-well plate with 5 mL of 1X PBS at room temperature.

2. Fix the organoid structures in Cultrex BME with 5 mL of 2% paraformaldehyde (PFA) + 0.1% glutaraldehyde (GA) in 1X PBS for 30 minutes at room temperature. NOTE: Occasionally PFA may cause depolymerization of the Cultrex BME. Addition of glutaraldehyde solves this issue, however, glutaraldehyde may produce higher auto-fluorescence, so it is important that the quenching step is optimized for each particular protocol.

3. Wash 3 times with 5 mL of 1X PBS for 10 minutes each wash to remove the fixing solution.

4. Carefully, take the organoid-BME domes with a scoop or spatula and place them in a 50 mL conical tube containing 20% sucrose in 1X PBS and leave the tube at 2–8 °C overnight or until the domes fall to the bottom of the tube (it may take up to 3 days).

5. Remove the domes from the sucrose solution and place in an embedding mold containing Optimal Cutting Temperature (OCT) compound. Try to remove as much of the sucrose as possible. Place several domes per mold. We recommend embedding at least six organoid-BME domes in each block.

6. Snap freeze and store at ≤ –70 °C.

7. Using a cryotome, cut the organoid block into cryosections. We recommend a 10 μm-thickness for organoid cryosections.

Immunostaining

1. Wash the slides once with 1X PBS for 15 seconds to remove the OCT compound. OPTIONAL: Use a hydrophobic marker to delimit the area around the organoids.

2. Quench the aldehyde groups produced after the fixing steps by incubating the slides with a 10 mM solution of NaBH₄ in 1X PBS twice for 5 minutes at room temperature each time. If auto-fluorescence is detected, increase the quenching washes. NOTE: A 15 minute incubation done twice with 0.2 M Glycine solution in 1X PBS can be used to quench the samples instead.

3. Wash 3 times with 1X PBS for 10 minutes each wash.

4. Permeabilize the tissues with 0.15% Triton + 1X PBS for 15 minutes at room temperature.

5. Wash 3 times with 1X PBS for 10 minutes each wash.

6. Block the slides with 3% BSA + 1X PBS (Blocking Solution) or 10% FBS + 1X PBS for 1–2 hours at room temperature.

7. Dilute the primary antibody in blocking solution to the desired final concentration. Tap the slides on the side to remove the blocking solution and, without washing, add 200–400 μL of the primary antibody to the organoids. Spread throughout the slide.

8. Incubate with the primary antibody overnight at 2–8 °C in a humidified chamber.

9. Wash the slides twice with 1X PBS for 15 seconds each wash.

10. Wash 3 times with 1X PBS for 10 minutes each wash.

11. Dilute the secondary antibodies in blocking solution to the desired final concentration. Add 200–400 μL of the secondary antibodies to the organoids.

12. Incubate at room temperature for 1.5–2 hours in a humidified chamber.

13. Wash 3 times with 1X PBS for 10 minutes each wash.

14. To counterstain, use a dilution of DAPI (Tocris Bioscience, Catalog # 5748) or Hoechst 33342 (Tocris Bioscience, Catalog # 5117) for nuclear staining, or Phalloidin to stain actin. All three counterstain compounds should be diluted in 1X PBS.

15. Wash once with 1X PBS for 10 minutes.

16. Rinse in distilled water, remove as much water as possible and mount using Fluorescent Mounting Medium (R&D Systems, Catalog # 4866-20).

17. Wait until the slides are dry and use an epi-fluorescence or a confocal microscope to visualize your samples.

Data Examples

Organoid Lumen Preservation

Both mouse small intestine and colon organoids develop a hollow lumen, as do other organoids, and it can be challenging to preserve these structures intact after fixing and cryosectioning. By following the protocol provided, it is possible to maintain the anatomy of the organoids as seen in Figures 3 and 4.

Expression of Intestinal Markers

Using this protocol, it is possible to analyze a variety of markers. In Figure 5, proliferating cells within crypt-like structures in mouse small intestine organoids are detected by staining for Ki-67. In Figure 6, production and secretion of Mucin 2 (MUC2), a mucus-forming glycoprotein secreted by goblet cells in the intestinal epithelium, is detected in mouse colon organoids.