TITLE: 622.2 URMC HTC Rapid  Clearing of Thick Human Lung Tissue Sections
AUTHORS: Cory  Poole, Gloria S Pryhuber

[DESCRIPTION]
Purpose and Scope of the Procedure:  
Provide a relatively rapid clearing of lung tissue sections up to 1 millimeter thick for immunofluorescence staining and imaging with confocal or multiphoton microscopy.
Current protocol is limited to tissue sections that are free from severe hemorrhage/thrombosis, containing mostly parenchyma, vessels, and smaller bronchioles. Times and solution volumes may need to be altered slightly if one wishes to clear sections containing a lot of cartilage and/or blood.


Scientific Principles: One of the major challenges in conventional brightfield, epifluorescence, and even confocal microscopy is the limitation in imaging depth. This is primarily due to the scattering and absorption of light within the sample of both the excitation light and the emission light leading to a drastic reduction in the image brightness and contrast when deeper than a 100µm. Clearing offers a way around this by both reducing the components that negatively impact the light and by matching the refractive index of the mounting medium to that of the components which make up the tissue, leading to an almost transparent appearance.

This protocol is a slight modification of a Clear Unobstructed Brain/Body Imaging Cocktail (CUBIC) protocol described by Kubota et al. developed originally to allow the analysis of cancer metastasis within the bodies of entire mice. By taking advantage of the fact lung tissue has plenty of open spaces and a very large surface area we can reduce the time in each of the solutions to allow complete clearing of the tissue sections to within just a few days.

References:
·Light Microscopy Methods and Protocols (2017). p. 74
·Kubota et al., (2017). Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution.Cell Reports 20, 236–250

[BEFORE_START]
Sections of lung between    and    should be cut and immediately processed or kept frozen (or cold, if cut with a vibratome) until processing begins. If not previously fixed with either formalin or paraformaldehyde then these tissue sections should be placed into fixative overnight before any mounting solution is washed away (if applicable).


[GUIDELINES]
This protocol is a slight modification of a Clear Unobstructed Brain/Body Imaging Cocktail (CUBIC) protocol described by Kubota et al. developed originally to allow the analysis of cancer metastasis within the bodies of entire mice. By taking advantage of the fact lung tissue has plenty of open spaces and a very large surface area we can reduce the time in each of the solutions to allow complete clearing of the tissue sections to within just a few days.

References:
·Kubota et al., (2017). Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution.Cell Reports 20, 236–250

When performing this protocol attempt to limit the generation of bubbles in the solutions that the sections will be incubating in. This can reduce the quality of the clearing, staining, and imaging as bubbles will keep the clearing or staining solutions from contacting the tissue. In addition bubbles are notorious for their negative effects on imaging due to the difference in refractive index.

When mounting the samples, work quickly to avoid drying of the CUBIC-R solution. Working in a more humid room may allow more time to work with mounting.

[STEPS]
SECTION: Preparing Reagents
1. 1X Phosphate Buffered Saline
Add 50 mL of 10X Phosphate Buffered Saline to 450 mL ultrapure water and mix the solution well. Store at room temperature.

100% CUBIC-L
Add 16 g of ultrapure water to a 50 mL conical followed by 2 g of Triton X-100. Swirl the tube until the Triton X-100 completely dissolves into solution. Next, in a fume hood, add 2 mL N-butyldiethanolamine to the solution and swirl until the solution is homogeneous. Place Parafilm around the cap to avoid any evaporation and store at room temperature.

50% CUBIC-L
In a separate 50 mL conical tube, add 5 mL of ultrapure water and 5 mL of 100% CUBIC-L solution. Swirl until the solution is properly mixed. Place Parafilm around the cap to avoid any evaporation and store at room temperature.

100%CUBIC-R
In a 50 mL conical tube add 5.0 g of ultrapure water followed by 6.0 g of N-methylnicotinamide. Close and shake the tube until all the N-methylnicotinamide is dissolved. Finally, add 9.0 g of Antipyrine to the tube and shake until completely dissolved. The solution should have a clear or slight yellow appearance to it. This solution is very susceptible to precipitation so Parafilm must be placed around the cap immediately after each use to avoid any evaporation. Store at room temperature.

50% CUBIC-R
In a separate 50 mL conical tube, add 5 mL of ultrapure water and 5 mL of 100% CUBIC-R solution. Swirl until the solution is properly mixed. Place Parafilm around the cap to avoid any evaporation and store at room temperature.

SECTION: Clearing the Tissue
2. Label the 50 mL conical tubes to be used with the appropriate section ID and fill each tube with 40 mL 1X Phosphate Buffered Saline (PBS). If the PBS is already a 1X solution you may ignore this step.

SECTION: Clearing the Tissue
3. Add sections in their respective 50 mL conical tube, placing the tube into a rack and onto the rotator at a low speed for 15 min. Use a transfer pipette to remove and add fresh PBS making sure to not damage the sections.

SECTION: Clearing the Tissue
4. After the 5th wash, add another 40 mL PBS and leave on the rotator at a low speed while preparing for the next step.

SECTION: Clearing the Tissue
5. Remove the PBS from the sample using a transfer pipette making sure to not damage the section in the process. Immediately add 5 mL of 50% CUBIC-L and gently swirl to verify the section is completely free floating. Place the 50mL conical tube into a rack. Repeat this step for all samples.

SECTION: Clearing the Tissue
6. When the previous step is completed, place the rack into a 37 10 incubator for a minimum of 180 min. Ideally the sections would be on a rotator at a low speed, but if this is unavailable then gently swirl the sections once or twice throughout the incubation if possible.

SECTION: Clearing the Tissue
7. Remove the sections from the incubator and remove the 50% CUBIC-L solution with a transfer pipette, ensuring there is no damage to any of the sections. 
SECTION: Clearing the Tissue
7.1. Dispose of the used 50% CUBIC-L solution into a properly labeled waste container.
SECTION: Clearing the Tissue
7.2.  Immediately after removing the 50% CUBIC-L solution from a section add5 mL of 100% CUBIC-L solution and giving the tube a gentle swirl and verifying the section is free floating. 

SECTION: Clearing the Tissue
7.3. Once each day, replace the used 100% CUBIC-L solution with fresh solution and place the samples back into the incubator. Add the removed 100% CUBIC-L to the waste container.
SECTION: Clearing the Tissue
7.4. Place the samples into a 37 °C  incubator. 
The samples will be in CUBIC-L solution for about 2880 min. This time may be longer or shorter depending on how much blood the tissue contains and how big the tissue section is.

SECTION: Clearing the Tissue
8. At the end of the incubation in CUBIC-L  remove the samples from the incubator and gently remove the 100% CUBIC-L solution.  

SECTION: Clearing the Tissue
8.1. Wash the samples with40 mL PBS on the rotator, 5 times for 15 min each. Moving the sections to a new container after a couple washes may help with the removal of the CUBIC-L solution.

SECTION: Clearing the Tissue
8.2. After the 5th wash replace with a fresh 40 mL PBS. At this stage you may temporarily store the sections overnight in a cold room.

SECTION: Clearing the Tissue
9. The samples are ready to be immunofluorescently stained. 
No more Triton X-100 should be used from this point on since it can have a negative impact on staining.
SECTION: Refractive Index Matching
10. 
 
After immunofluorescent staining is complete, from each section remove the last wash buffer with a transfer pipette and replace with2 mLof 50% CUBIC-R solution per section. The sections should be placed on a rotator at a low speed for at least 2 hours at room temperature and in the dark.

SECTION: Refractive Index Matching
11. Remove the 50% CUBIC-R and immediately replace with 2 mL of 100% CUBIC-R. Place these on the rotator at a low speed overnight (or a minimum of 240 min).

SECTION: Refractive Index Matching
11.1. Place the recovered 50% CUBIC-R into a 50 mL conical tube labeled for used 50% CUBIC-R solution to catch any sections that may be accidentally suctioned up with the solution.  Once it is determined that all the sections are present in their respective wells this used 50% CUBIC-R solution can be disposed of in a properly labeled waste container.

SECTION: Refractive Index Matching
12. Add 1 mL vacuum grease to a 3 mL syringe and attach the small cannula. In a 100 mm plate (or disposable glass slide if this is preferable), use this cannula to add a small ring of silicone grease a little larger than the tissue section to be mounted. Using a marker, make a label on the plate just next to the well that was previously created.

SECTION: Refractive Index Matching
13. Using 2 fine tipped forceps, gently grab a cleared tissue section by the 2 corners along the same edge to avoid folding of the section and gently place it in the previously made well. Smooth out the section as much as possible gently with the forceps after placing in the created well within the 100 mm plate.
SECTION: Refractive Index Matching
14. On top of this section add 1-2 drops of fresh 100% CUBIC-R solution. Gently work out any air bubbles formed during this process use the forceps. Work quickly as the CUBIC-R solution dries very fast and will begin to precipitate. 
SECTION: Refractive Index Matching
15. Gently place the glass coverslip over the section starting at an angle to avoid the formation of any air bubbles. Gently press the glass coverslip to ensure the glass and vacuum grease are sealed and no mounting medium is leaking. The section is now ready for imaging.
SECTION: Refractive Index Matching
16. Use a transfer pipet to move the used 100% CUBIC-R solution from the 6 well plate into a properly labeled waste container.
SECTION: Analysis of Results
17. A tissue section that has been perfectly cleared with this method should be almost completely transparent and around the same size as it was at the beginning of the clearing process. If the tissue section is in a clear bottom plate, position the plate over a sheet of paper with some text. ideally, the text will be perfectly readable through the tissue with little to no distortion. This serves as a good quick determination of the clearing quality. The true test is the quality of imaging with confocal or multiphoton microscopy in terms of imaging depth and clarity.