Perfusion and tissue preparation
Before intracardial perfusion, anesthetize the animals deeply with a combination of anesthetics MMF (1mL/100g of body mass for mice).
Transcardially perfuse the animals (e.g. using Leica perfusion one system at 100-125 mmHg pressure) with first heparinized (10U/mL of Heparin) 0.1 M PBS for 5-10 minutes then with 4% paraformaldehyde in 0.1 M PBS for 20 minutes at room temperature.
TIP: Skipping the Heparin when vasculature is labeled can increase the quality of labeling. For all perfusion steps, the needle should be placed and kept in the left ventricle of the heart and should not cross to the right side of the heart.
For whole-body clearing, remove the skin and carefully open the skull and vertebra without damaging the CNS tissue. At this point, the whole body clearing can be performed immediately, or the mouse body can be stored in 0.1M PBS at 4 °C up to 4 weeks. For clearing dissected organ, collect the tissues directly and post-fix them in 4% PFA for 1-2 days at 4°C. Wash the samples once in 0.1M PBS for 5 min before clearing.
Preparation of uDISCO solutions
- Prepare tert-butanol solutions with distilled water at 30 Vol%, 50 Vol%, 70 Vol%, 80 Vol%, 90 Vol%, 96 Vol% and 100 Vol% for gradient dehydration.
- Use Dichloromethane (DCM) as a pure solution, for delipidation step.
- Prepare refractive index matching solutions by mixing BABB (benzyl alcohol + benzyl benzoate 1:2, respectively) and diphenyl ether (DPE) at following ratio: BABB-D4, BABB:DPE at a ratio 4:1 (Vol/Vol); BABB-D10, BABB:DPE at a ratio 10:1 (Vol/Vol); BABB-D15, BABB:DPE at a ratio 15:1 (Vol/Vol). Add 0.4% Vol vitamin E into BABB-D solutions to scavenge the peroxides.
Tert-butanol is flammable, DCM is toxic and BABB-D components can cause skin irritation, therefore they should be handled carefully. Waste should be treated and discarded accordingly.
uDISCO passive clearing procedure for dissected organs
All incubation steps are performed in a fume hood with gentle rotation or shaking using 5 mL tubes (Eppendorf, 0030 119.401) for whole mouse brain or smaller samples, or using 80 ml glass chambers (omnilab, 5163279) for bigger samples such as rat tissues or whole brain + spinal cord. The samples are covered with aluminum foil to keep them in dark.
Incubate the fixed samples in 30 Vol%, 50 Vol%, 70 Vol%, 80 Vol%, 90 Vol%, 96 Vol% and 100% tert-butanol for 2-12 hours at 34-35°C (Table 1).
Incubate in DCM for 45-60 minutes at room temperature (small tissues such as mouse spinal cord or 1 mm-thick coronal slices can skip this step).
Incubate in BABB-D for 2-6 hours until the samples become optically transparent.
TIP: The higher amount of DPE in BABB yields better signal preservation (e.g. BABB-D15), while the lower amount of DPE in BABB results in higher transparency (e.g. BABB-D4). We recommend usage of BABB-D4 for small tissues e.g. spinal cord or tissue slices, and BABB-D15 for large tissues e.g. for rat brain clearing. For whole-body clearing with perfusion system, use BABB-D10.
Samples can be stored in BABB-D at room temperature in the dark for several weeks.
TIP: It is recommended to image sample as soon as possible to yield the best outcome.
uDISCO whole-body clearing procedure with perfusion system
Establish the transcardial-circulatory system as in Figure 1. Here, we used a peristaltic pump (Gilson, Peristaltic Pump MINIPULS 3) with one pumping channel (green dash line) and one recirculating channel (yellow dash line). All steps should be performed in a fume hood.
Connect the Viton reference tubing to the peristaltic pump.
TIP: As the clearing solutions can be corrosive to various tubing material, we recommend usage of Viton tubing (Gilson, F1817745).
Insert the tubing connectors (Omnilab, 5434482) (red arrow) at each end of the Viton tubing (blue arrow).
Connect the tubing connectors (red arrow) with additional PVC tubing (Omnilab, 5437920) for extension (orange arrow).
TIP: Transparent PVC tubing, which is compatible with clearing solutions, helps for checking unwanted air bubbles in the tubing system.
Cut the head part of the 1 ml syringe (Braun, 9166017V) as a connector (black arrow) and insert it into the outflow tubing of the pumping channel (orange arrow).
Connect the transcardiac perfusion needle (Leica, 39471024) (green arrow) for mouse or the thinnest perfusion needle (Leica, 39471022) without rubber head for rat with this connector. Subsequently, fix the inflow tubing of the recirculating channel in the glass chamber containing animal body ready for clearing.
TIP: Fix the tubing with tapes (any kind). Keep a certain height between the inflow tubing head and bottom of the glass chamber to ensure that the sample is covered by clearing solutions during clearing at all times.
Keep the inflow tubing of the pumping channel beneath the surface of the clearing solution and start the circulation until air bubbles are pushed out from the tubing system.
TIP: Avoid pumping air bubbles by keeping the tubing always immersed into solution while pumping.
Set the perfusion needle into the heart of the animal through the same pinhole made during perfusion and circulate the clearing solutions one by one as indicated in Table 1.
TIP 1: When starting the circulation, it might be visible that some ripples occur beside the right atrial appendage because the PBS in the sample is pushed into 30 Vol% tert-butanol solutions. This would be a good signal that the pumping is working appropriately. If not, try to change the angle of the perfusion needle to reach the best position and fix it with tapes. Because of the shrinkage during dehydration, the needle should stay in the heart without slipping out.
TIP 2: Stop the pump temporarily when changing the clearing solutions between steps. Collect the last solution back to the bottle by using a serological pipette and fill the glass chamber with the next solution quickly to minimize exposure to air. Because serological pipettes are not stable in BABB-D, use them only in the prior steps. Hold the inflow tubing ending of the pumping channel carefully to avoid any air bubble and put it beneath the surface of next solution. If there are large air bubbles within the tubing, they can be eliminated by a brief run of the pump in the reverse direction.
TIP 3: As the melting point of tert-butanol is 23 to 26 °C (close to room temperature), use a heating plate at 35-40 °C for 100% tert-butanol circulation steps to prevent the solution from solidification.
TIP 4: The amount of solutions for circulation depends on the capacity of the clearing chamber and the size of the animal that is being cleared. For example, for mice, a 400 ml capacity glass chamber with 300 ml working clearing solution and for rats, a 1000 ml capacity glass chamber with 800 ml working clearing solution would be sufficient.
TIP 5: For rat clearing, because PVC tubing is not resistant to DCM, the DCM step can be performed with gentle shaking to increase the efficiency.
TIP 6: The flowing rate was set at 8-10 ml/min for mouse clearing and 15-20 ml/min for rat clearing.
- At the final step, circulate BABB-D10 until full transparency is achieved (about 6-12 hours for mouse and ~24 hours for rat).
TIP: It is recommended to image sample as soon as possible to yield the best outcome.