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CLARITY Staining

Image the nervous system in three dimensions by making brain tissue optically transparent and macromolecule-permeable.
Last edited Wed 24 Jul 2019

CLARITY is a tissue-clearing method that transforms intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. CLARITY enables intact-tissue in situ hybridization, immunohistochemistry, and antibody labelling. This allows fine structural analysis of clinical samples, in a form suitable for probing the underpinnings of physiological function and disease.

Developed by the Chung Lab.

Materials Required

Reagents            

Beuthanasia-D

32% Paraformaldehyde (PFA)

40% ​Acrylamide solution

Azo-initiator

10X PBS

Ultrapure distilled water

Boric acid

Sodium dodecyl sulfate (SDS)

Lithium hydroxide monohydrate

N-methyl-D-glucamine

Diatrizoic acid

60% Iodixanol   

Triton-X (TX)      

Sodium azide    

1X PBS

Equipment

Transcardial perfusion of fixatives and hydrogel monomers

Dissection board (Styrofoam lid is fine)

20 ml syringes with luer lock ends

1 ml syringes

Winged infusion sets

Needles

Absorbent pads

50 mL Falcon tubes

Guillotine, for sacrificing larger animals

Surgical scissors

Fine scissors10

Hemostats

Forceps

Spatula

 

Hydrogel-tissue hybridization

Desiccator with 3-way stopcock

Vacuum pump

Compressed nitrogen tank

Compressed gas tank pressure regulator              

Teflon tape

3/8” tubing

3/8” to 1/4" barbed tubing connector

ETC clearing system

Buffer Filter with Light-Blocking Blue Bowl

Platinum wire with 0.5mm diameter

Bottle for Chamber fabrication

Nalgene Straight Side Jar – Poly, 32oz

Single barbed tube fitting (7/16” hex for 1/4” tubing)

Tube to tube coupling for 3/32” to 1/16” tubing 

3M Duo adhesive dispenser

3M Duo adhesive-mixing applicators

3M Duo adhesive cartridges

Sample holder

Bio-Rad HC PowerPac System

Banana to Large Alligator Test Lead Set

Clear 1/4" tubing

Clear 5/8” tubing

1/4" wye connector

4x Chemical resistant stopcock 1/4" to 1/4"

5/8" to 1/4" tubing connection

Elbow connection 1/4" male pipe to 1/4" barbed fitting

Elbow connection 1/4" barbed fitting

Rubber grounding plug

Magnetic water pump

Tissue

In principle, any tissue type from any animal of any age with or without fluorescence can be used. In the previous paper1, we demonstrated that CLARITY is compatible with the whole adult mouse brain, whole adult zebrafish brain, and extensively formalin-fixed post-mortem human brain section (without the perfusion step and further optimization in this case).

Tissues with strong fluorescent protein expression can undergo CLARITY processing described in this protocol and then be directly imaged; tissues without fluorescent proteins can be labelled with antibodies or RNA probes1 for subsequent imaging.

Stage 1 - Solution preparation

Steps

1

Keeping all reagents on ice, prepare a 10% stock solution of initiator solution by dissolving 1 g of azo-initiator in 10 mL UltraPure water.

  • Then prepare the following solution:
Solution Volume 

UltraPure water

26 mL

40% Acrylamide solution

4 mL

Initiator solution

1 mL

10X PBS

4 mL

32% PFA

5 mL

Stage 2 - Equipment setup

Steps

1

Mount the nitrogen tank with an appropriate tank bracket and attach the regulator to the tank outlet using Teflon tape if necessary to prevent leaking.

2

Run 3/8” tubing from the regulator outlet to the stopcock of the desiccator using a 3/8” to 1/4” barbed tube fitting.

3

Connect the vacuum pump to the desiccator by simply connecting the supplied tubing to the barbed fitting on the stopcock.

Stage 3 - Perfusion and Tissue Preparation

Steps

1

Make a fresh batch of hydrogel monomer solution, or thaw frozen stock solution at 4⁰C or on ice.

  • After the solution is completely thawed and transparent (but still ice-cold), gently invert to mix. 
  • Ensure no precipitation or bubbles are seen in the solution.
2

Deeply anesthetize an animal with beuthanasia-D (0.5 mL per 1 kg of body weight intraperitoneally).

  • Surgically open the chest cavity with a midline abdominal incision that bifurcates rostrally into a Y-shape. 
  • Punch a small hole in the right atrium and insert an injection needle into the left ventricle to allow perfusion.
3

Prepare two syringes filled with ice-cold PBS and hydrogel monomer solution, respectively, each with winged needle sets for each solution.

  • In the case of mice, perfuse first with 20 mL of ice-cold PBS at a rate of less than 5 mL/min, carefully take the needle out and perfuse with 20 mL of the ice-cold hydrogel monomer solution. Rats require about 200 mL of each solution at the rate of 20 mL/min.
  • Critical step: maintain a slow rate of perfusion. We found that injecting less than 5 mL per minute for both solutions in the case of mice yields better results. Use extreme caution not to introduce bubbles to the vasculature (especially when introducing needles), as this decreases the quality of perfusion.
4

Carefully harvest the organs of interest and place them immediately in a 50 mL conical tube containing 20 mL of the ice-cold hydrogel monomer solution for both post-fixation and even infiltration of monomers.

  • Keep this on ice until it can be transferred to a 4⁰C refrigerator.
5

Incubate the sample for one day at 4⁰C to allow for further distribution of monomer and initiator molecules throughout the tissue.

Stage 4 - Hydrogel tissue embedding

Steps

1

After the tissues have been allowed to incubate in the hydrogel monomer solution for one day, move the samples to 10 mL of fresh hydrogel monomer solution.

2

Place the conical tubes in a desiccation chamber on a tube rack and unscrew the caps about halfway.

3

Connect nitrogen gas and a vacuum pump to the desiccator via the three-way stopcock.

  • Open flow in all three directions and turn on the nitrogen gas.
  • Allow the gas to flow for about five seconds. 
4

Without turning off the nitrogen flow, turn on the vacuum pump and adjust the stopcock so that flow is only open to the desiccator and the vacuum pump.

  • Allow the vacuum pump to run for at least ten minutes.
5

Very slowly turn the stopcock so that flow is only open to the nitrogen gas and the desiccator, then turn off the vacuum pump.

  • Allow the desiccation chamber to fill with nitrogen gas.
6

Very quickly, lift the lid of the desiccator and tighten the caps of the conical tubes inside.

  • The nitrogen gas can now be shut off.
7

Gently shake the samples in a 37°C warm room for two hours.

  • This temperature will trigger radical initiation by the azo-initiator.
8

To remove unreacted PFA, wash the samples in 50 mL of clearing solution at 37°C for 24 hours, with gentle shaking.

  • Do this a total of three times.

Stage 5 - Electrophoretic tissue-clearing

At this point, you should have already constructed an ETC system as detailed in the section equipment setup.

Steps

1

Add the sample to the ETC chamber and close the lid.

  • . Connect any remaining unconnected tubing.
2

Fill the system with clearing solution by first filling the measurement reservoir and placing it on a surface a few inches higher than the level of the heat exchanger and pump.

  • This will allow the buffer to fill the tubing. 
  • Start the pump and add more clearing solution to the measurement reservoir as needed to fill the system.
3

Connect the electrodes to the lead cables and start the power supply.

  •  Use around 40 V.
4

Check the samples regularly to determine that the system is working properly and that clearing is progressing.

  • The entire process should take several days.
5

Remove the cleared samples from the ETC system and wash them twice with for 24 hours each.

6

Place the sample in a volume of optical clearing solution that is sufficient to cover the tissue completely and allow it to incubate for two days.

  • After the first day, move the sample to a container of fresh optical clearing solution.
7

To image the cleared sample, it must be mounted between a glass slide and a black Willco dish.

  • Roll up a piece of Blu-Tack adhesive into cylinder shapes of a thickness slightly more than the thickness of your sample.
  • Place them horizontally on the glass slide.
  • Press down the edge of Blu-Tack to close up the gap between the Blu-Tack adhesive and the glass slide (Shown in pictures).
8

Carefully place the sample in between the Blu-Tack pieces and add about 20 μL of optical clearing solution to the sample.

9

With the lipped side facing up, firmly press a Willco dish down onto the adhesive until it just comes into contact with the sample.

  • Using a pipette, add more optical clearing solution to the gaps between adhesive until the imaging chamber is filled.
10

KWIK-SIL is an adhesive that cures rapidly – carefully add it to the gaps between the Blu-Tack to build a wall and seal in the sample.

11

Cover this construction with aluminum foil and store it away safely to cure.

  •  After about 20 minutes, the sample is ready for imaging.