TITLE: Protocol for CUBIC Clearing and Whole Mount Imaging of Mouse Lung Lobes
AUTHORS: Justinn  Barr, Jamie   Verheyden, Xin      Sun

[DESCRIPTION]
This protocol is for Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) of mouse lung tissue for whole lobe imaging using Zeiss Lightsheet Imaging. 

All experimental procedures were performed in the American Association for Accreditation of Laboratory Animal Care (AAALAC)-certified laboratory animal facility at the University of California San Diego, following protocols approved by the institutional animal care and use committee (IACUC). The procedures should incorporate all local requirements for standards of animal experimentation.



[STEPS]
SECTION: Clearing preparations
1. 
Euthanize mice by CO2 inhalation.


SECTION: Clearing preparations
1.1. To clear blood from the lungs perfuse transcardially the animals with 1xPBS. Gravity inflate lungs with 4% paraformaldehyde (PFA) and fix overnight at 4 °C .
SECTION: Clearing preparations
1.2. Wash the lungs at least 3x for 10 minutes with 1xPBS rotating at Room temperature .
SECTION: Clearing preparations
1.3. Separate lobes to image them individually after cleaning.
SECTION: Clearing procedures
2. Rotate the lungs at Room temperature in CUBIC R1 buffer for at least 1 week.

Multiple lung lobes can be cleared in a 15 mL tube filled with R1 buffer.
If the buffer starts turning yellow or green add a fresh buffer to the sample.  
Clearing can be done at 4 °C  but will take longer.
 

SECTION: Clearing procedures
2.1. After 1 week move samples to 4 °C room to continue clearing until you are ready for imaging or to store the samples in CUBIC R1 buffer.

SECTION: Clearing Solutions
3. CUBIC buffers were prepared according to Muntifering et al., (2018)
SECTION: Clearing Solutions
4. CUBIC R1 - 500 g / ~420 mL 
SECTION: Clearing Solutions
4.1. Mix 125 g  of Urea (Sigma-Aldrich) and 175 mL H2O in a glass beaker.

SECTION: Clearing Solutions
4.2. Stir on a hot plate over low heat or place in a water bath, up to 56 °C , until the urea dissolves.
 


SECTION: Clearing Solutions
4.3. Add 123 g  (or 124 mL ) of Quadrol (Sigma-Aldrich).

 

SECTION: Clearing Solutions
4.4. Stir over low heat until the Quadrol dissolves.
SECTION: Clearing Solutions
4.5. Add 70 mL of TritonX-100 (Fisher, cat. no. BP151-500).

SECTION: Clearing Solutions
4.6. Remove from heat and stir until dissolved.
SECTION: Clearing Solutions
4.7. Store the solution sealed at Room temperature for approximately 1 month.

When the solution takes on a strong ammonia smell, it has expired.

If the temperature is too high when making the solution, the ammonia smell will be immediately present, and the solution should be discarded.

SECTION: Clearing Solutions
5. CUBIC R2  - 500 g / ~380 mL 
SECTION: Clearing Solutions
5.1. Mix 125 g  of Urea (Sigma-Aldrich) and 75 mL H2O in a glass beaker.
SECTION: Clearing Solutions
5.2. Stir on a hot plate over low heat or place in a water bath, up to 56 °C , until the urea dissolves.
 


 

SECTION: Clearing Solutions
5.3. Slowly add 250 g of sucrose (Sigma-Aldrich) with stirring over low heat.

SECTION: Clearing Solutions
5.4. Stir until dissolved using low heat. When dissolved, the solution will be extremely viscous.
SECTION: Clearing Solutions
5.5. Turn off heat and add 44.5 mL of Triethanolamine (TEA) with stirring.

SECTION: Clearing Solutions
5.6. Add 380 µL of TritonX-100 until well mixed.

SECTION: Clearing Solutions
5.7. Store the solution sealed at Room temperature for approximately 1 month.

When the solution takes on a strong ammonia smell, it has expired.

If the temperature is too high when making the solution, the ammonia smell will be immediately present, and the solution should be discarded.
SECTION: Light sheet imaging preparation
6. One day before imaging:
SECTION: Light sheet imaging preparation
6.1. Embed cleared lung sample in 3% low melt agarose.
SECTION: Light sheet imaging preparation
6.2. Once the agarose block has hardened, trim the block as small as possible for imaging. 
SECTION: Light sheet imaging preparation
6.3. If needed, attach staples with superglue for suspending in light sheet chamber.
SECTION: Light sheet imaging preparation
6.4. Store the agarose block with staples attached in R2 buffer overnight so that the agarose meets the refractive index of the CUBIC R2 buffer by the time of imaging.
SECTION: Imagining
7. Imaging was done with Zeiss Z.1 Lightsheet
SECTION: Imagining
7.1. Fill light sheet chamber with CUBIC R2 buffer.
SECTION: Imagining
7.2. Suspend the sample by attaching a magnet to the sample holder and using the magnet to hold the staples and agarose block.
SECTION: Imagining
7.3. To get the largest view of the sample, use a 2.5x objective with a large imaging chamber (Translucence Biosystems, Mesoscale Imaging System).
SECTION: Imagining
7.4. For long term storage place the sample back in the CUBIC R1 buffer. Do not leave the sample in the CUBIC R2 buffer for a long time.