Protocol for CUBIC Clearing and Whole Mount Imaging of Mouse Lung Lobes

Euthanize mice by CO₂ inhalation.

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 .

Wash the lungs at least 3x for 10 minutes with 1xPBS rotating at Room temperature .

Separate lobes to image them individually after cleaning.

Rotate the lungs at Room temperature in CUBIC R1 buffer for at least 1 week.

• Multiple lung lobes can be cleared in a 15mL 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.
  Note

  Clearing procedures were done according to Susaki et al., (2015)

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.

CUBIC buffers were prepared according to Muntifering et al., (2018) Muntifering et al., (2018)

CUBIC R1 - 500g / ~420mL

Mix 125g of Urea (Sigma-Aldrich) and 175mL H₂O in a glass beaker.

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

Add 123g (or 124mL ) of Quadrol (Sigma-Aldrich).

Stir over low heat until the Quadrol dissolves.

Add 70mL of TritonX-100 (Fisher, cat. no. BP151-500).

Remove from heat and stir until dissolved.

Store the solution sealed at 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.

CUBIC R2 - 500g / ~380mL

Mix 125g of Urea (Sigma-Aldrich) and 75mL H₂O in a glass beaker.

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

Slowly add 250g of sucrose (Sigma-Aldrich) with stirring over low heat.

Stir until dissolved using low heat. When dissolved, the solution will be extremely viscous.

Turn off heat and add 44.5mL of Triethanolamine (TEA) with stirring.

Add 380µL of TritonX-100 until well mixed.

Store the solution sealed at 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.

One day before imaging:

Embed cleared lung sample in 3% low melt agarose.

Once the agarose block has hardened, trim the block as small as possible for imaging.

If needed, attach staples with superglue for suspending in light sheet chamber.

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.

Imaging was done with Zeiss Z.1 Lightsheet

Fill light sheet chamber with CUBIC R2 buffer.

Suspend the sample by attaching a magnet to the sample holder and using the magnet to hold the staples and agarose block.

To get the largest view of the sample, use a 2.5x objective with a large imaging chamber (Translucence Biosystems, Mesoscale Imaging System).

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.