Dec 10, 2021

Public workspaceProtocol for CUBIC Clearing and Whole Mount Imaging of Mouse Lung Lobes

DOI
dx.doi.org/10.17504/protocols.io.b2gvqbw6
  • 1Department of Pediatrics, University of California, San Diego, CA 92093, USA;
  • 2Division of Biological Sciences, University of California, San Diego, CA 92093, USA
  • SPARC
    Tech. support email: info@neuinfo.org
Marlena S Pela
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DOI: dx.doi.org/10.17504/protocols.io.b2gvqbw6
Protocol Citation: Justinn Barr, Jamie Verheyden, Xin Sun 2021. Protocol for CUBIC Clearing and Whole Mount Imaging of Mouse Lung Lobes. protocols.io https://dx.doi.org/10.17504/protocols.io.b2gvqbw6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 30, 2021
Last Modified: December 10, 2021
Protocol Integer ID: 55541
Abstract
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.


Materials
Subjects:

ABCD
SpeciesStrainRRID for strainSupplier
Mus musculusVglut2-ires-creIMSR_JAX:028863The Jackson Laboratory
Mus musculusRosa-lxl-tdTomatoIMSR_JAX:007914The Jackson Laboratory
Mus musculusNkx2-1 GFPMMRRC_066764-JAXGift from Dr. D. Kotton and Dr. L. Ikonomou
The Cre lines used in the experiments were kept in B6 background. Cre males were mated to B6 wild-type females and both male and female mice were used in the experiment.
CUBIC R1 buffer:

CUBIC R2 buffer:

Light Sheet Microscopy:

Clearing preparations
Clearing preparations

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 Temperature4 °C .
Wash the lungs at least 3x for 10 minutes with 1xPBS rotating at TemperatureRoom temperature .
Separate lobes to image them individually after cleaning.
Clearing procedures
Clearing procedures
Rotate the lungs at TemperatureRoom temperature in CUBIC R1 buffer for at least 1 week.

  • Multiple lung lobes can be cleared in a Amount15 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 Temperature4 °C but will take longer.
Note
Clearing procedures were done according to Susaki et al., (2015)

After 1 week move samples to Temperature4 °C room to continue clearing until you are ready for imaging or to store the samples in CUBIC R1 buffer.

Clearing Solutions
Clearing Solutions
CUBIC buffers were prepared according to Muntifering et al., (2018)
CUBIC R1 - Amount500 g / ~Amount420 mL
Mix Amount125 g of Urea (Sigma-Aldrich) and Amount175 mL H2O in a glass beaker.

Stir on a hot plate over low heat or place in a water bath, up to Temperature56 °C , until the urea dissolves.
Note
Allowing the mixture to reach a temperature of up to 56 degrees, will facilitate
other components going into solution, but this step is not necessary.


Add Amount123 g (or Amount124 mL ) of Quadrol (Sigma-Aldrich).

Note
Quadrol is very viscous, therefore, it should be weighed directly into the urea solution. If the volume must be measured by volume, heat the Quadrol to Temperature56 °C in a water bath prior to pouring.

Stir over low heat until the Quadrol dissolves.
Add Amount70 mL of TritonX-100 (Fisher, cat. no. BP151-500).

Remove from heat and stir until dissolved.
  • Store the solution sealed at TemperatureRoom 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.

CUBIC R2 - Amount500 g / ~Amount380 mL
Mix Amount125 g of Urea (Sigma-Aldrich) and Amount75 mL H2O in a glass beaker.
Stir on a hot plate over low heat or place in a water bath, up to Temperature56 °C , until the urea dissolves.
Note
Allowing the mixture to reach a temperature of up to 56 degrees, will facilitate
other components going into solution, but this step is not necessary.


Note
The container should remain loosely capped to limit evaporation.

Slowly add Amount250 g 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 Amount44.5 mL of Triethanolamine (TEA) with stirring.

Add Amount380 µL of TritonX-100 until well mixed.

  • Store the solution sealed at TemperatureRoom 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.
Light sheet imaging preparation
Light sheet imaging preparation
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.
Imagining
Imagining
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.