Immunostaining and clearing of primary human tumors and patient-derived xenografts for 3D imaging analysis

• Always freshly prepare reagents. Failure to do so may lead to suboptimal results.

• 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.

1. 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.
2. 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.
3. Adjust the pH to 7.2–7.4 with HCl and NaOH.
4. Adjust the volume to 500 mL with PBS.
5. Filter the solution using a 0.22 µm filter.
6. 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.

• 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 1× Antibody Staining Solution.

• The following solutions are needed: 
  • Ethanol 30%/Tween® 20 
  • Ethanol 50%/Tween® 20 
  • Ethanol 70%/Tween® 20
  • Ethanol 90%/Tween® 20
  • Absolute (≥99.8%) ethanol/Tween® 20
• Prepare ethanol dilutions of 30%, 50%, 70%, and 90% 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%. 

• Prepare depigmentation solution by dissolving 7.5 g/L carbamide peroxide in ethanol (e.g. 750 mg in 100 mL). Use absolute ethanol (≥99.8%) to prepare the solution.
  ▲ Note: Carbamide peroxide is hazardous. Refer to the manufacturer’s website for the safety data sheet.
  ▲ Note: Store at 2–8°C and protected from light. For long-term storage use a glass bottle.

• 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 tweezers to handle the tissue.

1. Cut tumor tissue in pieces of 5×5×5 mm³.
2. Transfer samples into a tube containing PFA buffer at 2–8 °C.
   ▲ Note: Make sure all samples are fully immersed in PFA buffer.
3. Incubate overnight at 2–8 °C. 
   ▲ 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.
   ▲ Note: In order to avoid autofluorescence, do not extend the incubation time.
4. Wash samples 3× in PBS. 
   ▲ Note: If not immediately processed for clearing, samples can be stored in PBS/azide buffer at 2–8 °C for several weeks.

1. Add 5 mL of Permeabilization Solution of the MACS Clearing Kit to a 5 mL tube.
2. Transfer one sample into the tube.
   ▲ Note: Place only one sample in each tube to allow sufficient interaction of chemicals with the sample. 
3. Incubate for 24 hours at room temperature (20–25 °C) under slow continuous rotation using the MACSmix Tube Rotator.
4. After incubation, in case of blood-rich samples proceed optionally with 2.3 Depigmentation. If depigmentation is not needed, immediately proceed to section 2.4 Immunostaining.
   ▲ Note: Depigmentation is strongly recommended for blood-rich samples.

2.3.1 Dehydration

• Perform all incubation steps protected from light.
• Dehydration can be performed in the same 5 mL tube already containing the sample from 2.2 Permeabilization. Remove solvents as neatly as possible from the tube before adding the ethanol dilutions.

1. Discard Permeabilization Solution from 2.2 Permeabilization.
2. Dehydrate sample by incubation in a series of ethanol dilutions (one tumor piece per tube), at room temperature (20–25 °C) under slow, continuous rotation using the MACSmix Tube Rotator:
   1. Incubation in 30% ethanol/Tween® 20 for 4 hours
   2. Incubation in 50% ethanol/Tween® 20 for 4 hours
   3. Incubation in 70% ethanol/Tween® 20 overnight
   4. Incubation in 90% ethanol/Tween® 20 for 4 hours
   5. Incubation in absolute (≥99.8%) ethanol/Tween® 20 for 4 hours

2.3.2 Depigmentation

• Use a new tube for the depigmentation step. Transfer dehydrated sample into the tube using tweezers.

1. Add 10 mL of depigmentation solution to a 50 mL tube.
2. Transfer dehydrated sample pieces into the tube with depigmentation solution.
3. Incubate for 16 hours overnight at 4 °C at 400 rpm using the Eppendorf Thermomixer® with Eppendorf SmartBlock™ 50 mL.

2.3.3 Rehydration

• Perform all incubation steps protected from light.

1. Transfer the sample to a new 5 mL tube. Rehydration steps can be performed in the same 5 mL tube. Remove solvents as neatly as possible from the tube before adding the ethanol dilutions.
2. Rehydrate sample by incubation in a series of ethanol dilutions (one tumor piece per tube), at room temperature (20–25 °C) under slow, continuous rotation using the MACSmix Tube Rotator:
   1. Incubation in 90% ethanol/Tween® 20 for 2 hours
   2. Incubation in 70% ethanol/Tween® 20 for 2 hours
   3. Incubation in 50% ethanol/Tween® 20 for 2 hours
   4. Incubation in 30% ethanol/Tween® 20 for 2 hours
   5. Incubation in PBS overnight

• 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.

1. Dilute the primary antibody in a final volume of 0.5 mL of 1× Antibody Staining Solution. Use 1.5 mL tubes to prepare the dilution.
2. Transfer one sample per tube containing the antibody dilution.
3. Incubate sample with gentle shaking using a horizontal shaker. When using Miltenyi Biotec antibodies, incubate for 7 days at 37 °C.
4. Washing step 1: Add 5 mL of 1× Antibody Staining Solution into a 5 mL tube. Transfer sample into this tube. Incubate sample under slow, continuous rotation for 4 hours at room temperature (20–25 °C) using the MACSmix Tube Rotator.
5. Washing step 2: Discard 1× Antibody Staining Solution and repeat step 4.
6. Washing step 3: Perform one additional washing step with 5 mL of 1× Antibody Staining Solution. Incubate overnight at room temperature (20–25 °C) under slow, continuous rotation using the MACSmix Tube Rotator.
7. If working with fluorochrome-conjugated primary antibodies, discard 1× Antibody Staining Solution and continue with section 2.5 Dehydration. 
8. If performing indirect staining, follow steps 9–11. 
9. Dilute secondary antibody in a final volume of 1.5 mL of 1× Antibody Staining Solution. Use a new tube to prepare the dilution.
   ▲ Note: Refer to the manufacturer's recommendations for antibody dilution.
10. Incubate sample in secondary antibody dilution with gentle shaking using a horizontal shaker.
    ▲ Note: Refer to the manufacturer's recommendations for incubation temperature and duration of incubation.
11. Proceed with the washing steps above (steps 4–6).

• Perform all incubation steps protected from light.
• 5 mL tubes must be completely filled with the corresponding ethanol dilution.
• Dehydrate samples by incubation in a series of ethanol dilutions in 5 mL amber tubes at room temperature (20–25 °C) under slow, continuous rotation using the MACSmix Tube Rotator.
  ▲ Note: All 4 hour incubation steps can be extended to overnight incubation.

1. Incubation in 30% ethanol/Tween® 20 for 4 hours
2. Incubation in 50% ethanol/Tween® 20 for 4 hours
3. Incubation in 70% ethanol/Tween® 20 overnight
4. Incubation in 90% ethanol/Tween® 20 for 4 hours
5. Incubation in absolute (≥99.8%) ethanol/Tween® 20 for 4 hours
6. Incubation in absolute (≥99.8%) ethanol/Tween® 20 overnight
   ▲ Note: Replace absolute (≥99.8%)  ethanol/Tween® 20 from step 5 with fresh one and incubate overnight. The tissue can then be stored long-term in absolute (≥99.8%) ethanol/Tween® 20.

• Perform all incubation steps protected from light.

1. Discard ethanol dilution and add 2.5 mL of Clearing Solution of the MACS Clearing Kit into the same 5 mL tube. 
   ▲ Note: Always use polypropylene tubes to incubate sample with the Clearing Solution of the MACS Clearing Kit.
2. Incubate at room temperature (20–25 °C) under slow continuous rotation for 6 hours using the MACSmix Tube Rotator.
3. Replace Clearing Solution with fresh Clearing Solution after 6 hours and continue incubation overnight.
4. Discard Clearing Solution and continue with downstream image acquisition and analysis.
   ▲ Note: Cleared samples can be stored in 5 mL polypropylene or glass tubes filled with MACS Imaging Solution  at room temperature for several months.
   ▲ Note: Do not store cleared samples at 2–8 °C, otherwise freezing artifacts and tissue damage may occur.
5. (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.