Immunostaining and clearing of organoids and spheroids for 3D imaging analysis
This application protocol describes step-by-step the immunostaining and clearing of organoids and spheroids using the MACS® Clearing Kit for downstream 3D imaging analysis. The MACS Clearing Kit enables the immunostaining and clearing of up to 60 samples of <2 mm in diameter.
Protocol
- Always freshly prepare reagents. Failure to do so may lead to suboptimal results.
Preparation of PFA buffer
- All steps that include paraformaldehyde (PFA) must be performed under a fume hood.
- The protocol describes the preparation of 500 mL of 4% PFA buffer. For higher or lower volumes scale the amounts of the components accordingly.
- Do not use ready-to-use PFA solutions.
- Add 20 g of PFA powder to 400 mL of PBS and stir the mixture at 60 °C under a fume hood until it is dissolved
▲ Note: Do not boil!
▲ Note: PFA powder does not dissolve instantly. - Add 0.2–0.4 g of NaOH and continue stirring at 60 °C until the solution clears up. This may take 2–3 hours.
- Adjust the pH to 7.2–7.4 with HCl and NaOH.
- Adjust the volume to 500 mL with PBS.
- Filter the solution using a 0.22 µm filter.
- PFA buffer can be stored at 4 °C for one week. For long-term storage prepare aliquots and store at – 20 °C. Avoid repeated freeze/thaw cycles.
Preparation of 1× Antibody Staining Solution
- If precipitates occur due to storage of the 10× Antibody Staining Solution at low temperature, mix well before preparation of 1× Antibody Staining Solution.
- 10× Antibody Staining Solution needs to be diluted 1:10 with sterile distilled water before use, e.g., by diluting 1 mL of 10× Antibody Staining Solution with 9 mL of sterile distilled water. Adjust the volume of 1× Antibody Staining Solution according to the number of samples to be cleared.
- Always use freshly prepared diluted 1× Antibody Staining Solution.
Preparation of ethanol/Tween® 20 dilutions for dehydration
- The following solutions are needed:
- Ethanol 50%/Tween® 20
- Ethanol 70%/Tween® 20
- Absolute (≥99.8%) ethanol/Tween® 20
- Prepare ethanol dilutions of 50% and 70% in water. Use absolute ethanol (≥99.8%) and sterile distilled water to prepare the dilutions. Add Tween® 20 to each dilution for a final concentration of 2%.
▲ Note: Make sure to use fresh absolute ethanol (≥99.8%) that does not contain water residues.
Preparation of Clearing Solution
- Clearing Solution is ready-to-use, but may freeze when stored at 2–8 °C. Therefore, it is recommended to prepare aliquots and store at 2–8 °C. Take out aliquots to room temperature (20– 25 °C) before proceeding to clearing of samples.
▲ Note: Use polypropylene tubes for preparation of aliquots. Other plastics might not be compatible with the Clearing Solution.
- For details on the reagents of the MACS Clearing Kit refer to the respective data sheet.
- Use cut pipette tip and tweezers to handle the samples.
2.1 Fixation
- Generate samples using standard protocols.
- Gently transfer samples into a tube using wide or cut pipette tips.
- Wash samples 3× in PBS at 2–8 °C to remove residual medium.
- Discard PBS and add PFA buffer
▲ Note: Make sure all samples are fully immersed in PFA buffer.
▲ Note: It is recommended to incubate the samples under slow, continuous rotation using the MACSmix™ Tube Rotator in a PFA volume that is at least 10 times higher than the volume of the samples. - Incubate for 20 minutes at room temperature (20–25 °C).
▲ Note: In order to avoid autofluorescence, do not extend the incubation time. - Wash samples 3× in PBS.
▲ Note: If not immediately processed for clearing, sample can be stored in PBS/azide at 2–8 °C for several weeks.
2.2 Permeabilization
- Incubate up to three samples in 0.5 mL Permeabilization Solution in 0.5 mL tubes for at least 6 hours at room temperature (20–25 °C) under slow, continuous rotation using the MACSmix Tube Rotator.
- After incubation discard Permeabilization Solution and immediately proceed to section 2.3
Immunostaining and washing.
2.3 Immunostaining and washing
- Perform all incubation steps protected from light.
- The following protocol has been optimized for fluorochrome-conjugated primary antibodies from Miltenyi Biotec. Refer to the respective Miltenyi Biotec antibody conjugate data sheet or product page for the recommended dilution. When using other antibodies refer to the manufacturer´s recommendations for antibody dilution, incubation temperature, and duration of incubation.
- Dilute the primary antibody in a final volume of 0.4 mL of 1× Antibody Staining Solution.
- Add antibody dilution into the 0.5 mL tube containing up to three permeabilized samples.
- Incubate with gentle shaking using the MACSmix Tube Rotator. When using Miltenyi Biotec antibodies, incubate samples for 40 hours at 37 °C.
▲ Note: For smaller samples (<200 µm in diameter) incubation time can be reduced to overnight. - Washing step 1: Discard the solution and add 0.5 mL of 1× Antibody Staining Solution into the 0.5 mL tube.
- Washing step 2: Incubate sample under slow, continuous rotation for 30 minutes at room temperature (20–25 °C) using the MACSmix Tube Rotator.
- Washing step 3: Repeat steps 4 and 5 five times.
- If working with fluorochrome-conjugated primary antibodies, continue with step 2.4 Embedding. If performing indirect staining follow steps 8–10.
- Dilute secondary antibody in a final volume of 0.4 mL of 1× Antibody Staining Solution.
Remove 1× Antibody Staining Solution and add secondary antibody dilution into the 0.5 mL tube containing the samples.
▲ Note: Refer to the manufacturer's recommendations for antibody dilution. - Incubate sample in secondary antibody dilution with gentle shaking using the MACSmix Tube Rotator.
▲ Note: Refer to the manufacturer's recommendations for incubation temperature and duration of incubation. - Proceed with washing steps above (steps 4-6).
2.4 Embedding
- Perform all incubation steps protected from light.
- For gel preparation dissolve 1.5% agarose in distilled water or 1% Phytagel™ in tap water by heating it up in a microwave.
- Gently transfer samples to a cryomold using wide or cut pipette tips. Place them in different corners of the cryomold to facilitate later cutting.
- Remove residual 1× Antibody Staining Solution using a pipette.
- Add 1 mL liquid gel to the cryomold on top of the samples.
▲ Note: Make sure that the liquid gel is not too hot. The glass bottle for gel preparation should be touchable by hand. - Wait 10–20 minutes until the gel solidifies.
- Cut the gel block into approximately 5×5 mm2 pieces, each containing one sample.
▲ Note: Each sample should be located in one corner of the gel cube to obtain optimal imaging conditions.
▲ Note: After clearing the organoid or spheroid will not be visible inside the cube anymore. Consider marking the cube to indicate the location of the sample within the cube, e.g., by cutting the edge of the cube that is opposite to the sample location.
2.5 Dehydration
- Perform all incubation steps protected from light.
- 5 mL tubes must be completely filled with corresponding ethanol/Tween® 20 dilution.
- Dehydrate up to three embedded samples with a series of ethanol dilutions in a 5 mL tube at room temperature (20–25 °C) under slow, continuous rotation using the MACSmix Tube Rotator.
▲ Note: All 2 hour incubation steps can be extended to overnight incubation.
- Incubation in 50% ethanol/ Tween® 20 for 2 hours
- Incubation in 70% ethanol/ Tween® 20 for 2 hours
- Incubation in absolute (≥99.8%) ethanol/Tween® 20 overnight
2.6 Clearing
- Perform all incubation steps protected from light.
- Add 2.5 mL of Clearing Solution of the MACS Clearing Kit into a 5 mL amber polypropylene tube.
▲ Note: Always use polypropylene tubes to incubate sample with the Clearing Solution of the MACS® Clearing Kit. - Transfer up to three samples into this tube.
- Incubate at room temperature (20–25 °C) under slow continuous rotation for 3 hours using the MACSmix Tube Rotator.
- Replace Clearing Solution with fresh Clearing Solution after 3 hours and continue incubation for additional 3 hours.
- Discard Clearing Solution and continue with downstream image acquisition and analysis.
▲ Note: Cleared samples can be stored at room temperature in 5 mL polypropylene or glass tubes filled with MACS Imaging Solution for several months.
▲ Note: Do not store cleared sample at 2–8 °C, otherwise freezing artefacts and tissue damage may occur. - (Optional) If image acquisition is performed immediately after clearing, transfer cleared samples into the imaging chamber of a light sheet microscope (e.g. Ultramicroscope II or Ultramicroscope Blaze). Fill imaging chamber with MACS Imaging Solution and proceed with image acquisition.
Materials
- MACS® Clearing Kit (# 130-126-719)
- MACSmix™ Tube Rotator (# 130-090-753)
- MACS Imaging Solution (# 130-126-335)
- Distilled water or tap water
- Tween® 20
- Absolute ethanol (≥99.8%)
- Phosphate-buffered saline (PBS), pH 7.4
- (Optional) PBS/azide buffer (PBS containing 0.02% sodium azide)
- PBS/EDTA buffer (PBS, pH 7.4, containing 5 mM EDTA)
- Paraformaldehyde (PFA) powder
- Low melting agarose, agarose or Phytagel™ (Sigma-Aldrich)
- Cryomolds
- Scalpel
- 0.5 mL, 5 mL, 15 mL, and 50 mL tubes with lid
- 5 mL amber polypropylene tubes with lid
- Microwave
For more information about light sheet fluorescence microscopy refer to Light sheet microscopy.
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