Immunostaining and clearing of mouse brain hemispheres 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. Store PFA buffer 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 from the MACS Clearing Kit 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 within one or two days before use to ensure the absence of residual water.
• Prepare absolute ethanol (≥99.8%) using activated molecular sieves (3Å; if the ethanol bottle is freshly opened, steps 1 to 7 are optional).
• Prepare ethanol dilutions of 30%, 50%, 70%, and 90% in water. Use absolute ethanol (≥99.8%) and sterile distilled water to prepare the dilutions. 

1. Cover the bottom of a heat-resistant glass petri dish with molecular sieves.
2. Heat molecular sieves in microwave at 750 W for 2 minutes.
3. Mix the sieves before heating again at 750 W for 2 minutes.
4. Repeat heating until no liquid vaporizes anymore and sieves are completely dry.
5. Let sieves cool down.
6. Transfer sieves into a bottle and add absolute ethanol.
7. Incubate the sieves for 1–2 days to absorb residual water.
8. Filter the needed volume of absolute ethanol from the previous step using a 0.22 µm filter and use sterile distilled water to make the respective (30%, 50%, 70%, 90%) dilutions.
9. Add Tween® 20 to each dilution to a final concentration of 2% (v/v).

• 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. Bring 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 samples.

2.1.1 Fixation of neonatal mouse brain hemispheres (from mice of age ≤P7)

1. Extract sample and collect it in a tube containing PBS/EDTA buffer at 2–8 °C.
2. Transfer brain immediately into a new tube containing PFA buffer at 2–8 °C.
   ▲ Note: It is possible to fix more than one mouse brain per tube. Make sure brains are fully
   immersed in PFA buffer.
3. Incubate overnight at 2–8 °C.
   ▲ Note: In order to avoid autofluorescence, do not extend the incubation time.
4. Wash sample 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.

2.1.2 Fixation of adult mouse brain hemispheres (from mice of age >P7)

1. For subsequent clearing of adult mouse brain hemispheres, it is highly recommended to perfuse mice transcardially. Use PFA buffer for transcardial perfusion.
2. Extract sample after perfusion and collect it in a tube containing PBS/EDTA buffer at 2–8 °C.
3. Transfer sample into a new tube containing PFA buffer at 2–8 °C.
   ▲ Note: It is possible to fix more than one mouse brain per tube. Make sure brains are fully immersed in PFA buffer.
4. Incubate for 2 hours at 2–8 °C.
   ▲ Note: In order to avoid autofluorescence, do not extend the incubation time.
5. Wash sample 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. Cut the brain into two hemispheres using a scalpel.
2. Add 5 mL of Permeabilization Solution of the MACS Clearing Kit into a 5 mL tube.
3. Transfer one brain hemisphere into the tube.
4. Incubate for at least 24 hours at room temperature (20–25 °C) under slow continuous rotation using the MACSmix™ Tube Rotator.
5. After incubation discard Permeabilization Solution and immediately proceed to section 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 data sheet or product page for recommended dilution. When using other antibodies than from Miltenyi Biotec, refer to the manufacturer´s recommendations for antibody dilution, incubation temperature, and duration of incubation.

1. Dilute the primary antibody to a final volume of 1.5 mL of 1× Antibody Staining Solution. Use a 24-well plate to prepare the dilution.
2. Transfer the sample into the 24-well plate containing the antibody dilution.
3. Carefully seal the plate with PARAFILM® to prevent evaporation.
4. Incubate the sample with gentle shaking using a horizontal shaker. When using Miltenyi Biotec antibodies, incubate the sample for 7 days at 37 °C.
5. Add 5 mL of 1× Antibody Staining Solution into a 5 mL tube.
6. Transfer the sample into this tube.
7. Washing step 1: To remove unbound antibody, incubate sample under slow, continuous rotation for 4 hours at room temperature (20–25 °C) using the MACSmix Tube Rotator.
8. Washing step 2: Discard 1× Antibody Staining Solution and repeat steps 5–7.
9. 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.
10. If working with fluorochrome-conjugated primary antibodies, discard 1× Antibody Staining Solution and continue with section 2.4 Dehydration.
11. If performing indirect staining, follow steps 12–14.
12. Dilute secondary antibody in a final volume of 1.5 mL of 1× Antibody Staining Solution. Use a new 24-well plate to prepare the dilution.
    ▲ Note: Refer to the manufacturer's recommendations for antibody dilution.
13. Incubate brain hemispheres 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.
14. Proceed with the three washing steps above (steps 5–9).

• For preparation of ethanol series refer to section 1. Reagent preparation.
• When working with mouse brain derived from ≤P7 mice all following incubation steps are performed at room temperature (20–25 °C). When working with mouse brain tissue derived from >P7 mice it is recommended to adapt the temperature during dehydration to 28 °C by using an Eppendorf ThermoMixer® C with Eppendorf SmartBlock™ 50 mL at a rotational speed of 500 rpm or a shaking water bath.

2.4.1 Dehydration of mouse brain hemispheres from ≤P7 mice

• Perform all incubation steps protected from light.
• 15 mL tubes must be completely filled with the corresponding ethanol dilution.
• Always use a new tube for each dehydration step. Transfer the sample into the tube using tweezers.
• Dehydrate neonatal brain hemispheres by incubation in a series of ethanol dilutions in 15 mL tubes (one hemisphere per tube) at room temperature (20–25 °C) under slow, continuous rotation using the MACSmix Tube Rotator.
• 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.

2.4.2 Dehydration of mouse brain hemispheres from >P7 mice

• Perform all incubation steps protected from light.
• 50 mL tubes must be completely filled with the corresponding ethanol dilution.
• Always use a new tube for each dehydration step. Transfer the sample into the tube using tweezers.
• Dehydrate sample with a series of ethanol dilutions in 50 mL tubes (one hemisphere per tube) at 28 °C using the Eppendorf ThermoMixer® C with Eppendorf SmartBlock™ 50 mL at a rotational speed of 500 rpm.
  ▲ Note: The Eppendorf ThermoMixer® C with Eppendorf SmartBlock™ 50 mL is recommended for dehydration. Alternatively, a shaking water bath can be used at 28 °C water temperature.
• 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. Add 5 mL of Clearing Solution of the MACS Clearing Kit into a 5 mL amber polypropylene tube.
   ▲ Note: Always use polypropylene tubes to incubate samples with the Clearing Solution of the MACS Clearing Kit.
2. Transfer one brain hemisphere into this tube.
   ▲ Note: Make sure the tube is completely filled with Clearing Solution. Add additional Clearing Solution if necessary to fill up the tube completely.
3. Incubate at room temperature (20–25 °C) under slow continuous rotation for 6 hours using the MACSmix Tube Rotator.
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 artefacts 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.