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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 or NP-40

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

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.

Solution
Volume (mL)
UltraPure water
26
40% Acrylamide solution
4
Initiator solution
1
10X PBS
4
32% PFA
5
Make sure to keep all reagents and the final solution on ice at all times. The hydrogel polymerization reaction is triggered by heat.
For each tissue sample being processed, 80 mL will be needed. Always add water first to keep the other components dilute, and add the reagents in the order listed here. The solution can be stored at -20°C indefinitely.

Steps

Adjust the following solution to pH 8.5 using boric acid.

Reagent
Concentration (mM)
Lithium hydroxide monohydrate
20
SDS
200

Steps

Make a solution consisting of 0.1% Triton-X (0.1%% NP-40 can be used instead) and 0.1% Sodium Azide using 1X PBS.

Reagent
Volume
1X PBS
500 mL
TX
500 µL
Sodium azide
500 mg

Steps

This solution consists of 23.5% (w/v) N-methyl-D-glucamine, 29.4% (w/v) diatrizoic acid, and 32.4% (w/v) iodixanol in water.

Reagent
Volume
47% Iodixanol solution in water*
10 mL
N-methyl-D-glucamine
3.39 g
Diatrizoic acid
4.24 g

*To create this, add approximately 2.75 mL water to every 10 mL of 60% iodixanol solution.

Do not use heat when mixing the solution, as this will cause a color change.

Use Teflon tape to increase the security of the bottle’s seal; parafilm can be used around the cap.

It may be necessary to use a 60% iodixanol solution (see reagents list) rather than iodixanol powder, as it is not cheaply available.

Stage 2 - Equipment setup

Steps

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.

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

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

Steps

Create the measurement reservoir in a similar manner using a 32 oz Nalgene bottle and 1/4" barbed elbow connectors.

Be sure to place the connections on opposite sides of the bottle, angling them slightly to maximize mixing in the chamber.

Apply epoxy to both the inside and outside parts of the connection and allow to dry overnight.

Don’t allow any epoxy to enter the tubing connectors, as this will impede flow within the system.

Create two more holes in the lid of the bottle, large enough for insertion of a pH probe and a thermometer for data acquisition.

Create a heat exchange module by measuring out two pieces of around 2 ft of 1/4" tubing.

Connect these to the system in parallel using wye connectors and submerge in water.

Connect the water filter to the system using 1/4" male pipe thread to 1/4" tubing elbow connectors.

Tube all the components of the system together using 1/4" tubing, though 5/8” tubing will be needed for the pump connection.

Placing the measurement reservoir direction after the ETC chamber allows for direct readouts of the temperature and pH as they are in the ETC chamber. It is also important that the reservoir is only separated from the pump inlet by tubing, as the reservoir is necessary to start the system. If desired, drain valves can be created using wye connectors and stopcocks and placed between any elements of the system.

When connecting the system with 1/4" tubing, 1/4" stopcocks should be added to the system on either side of the ETC chamber, so that it can periodically be isolated from the system to check the samples without draining the entire system.

Stage 3 - Perfusion and tissue preparation

Steps

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

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

Experiments involving animals must be conducted in accordance with governmental and institutional regulations. Animals must be fully anesthetized before making incisions: deep anesthesia can be confirmed by the absence of corneal reflex (eye blink) or by any other overt signs of response to physical stimuli.

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

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.

Always keep the temperature low to prevent thermal initiation of the hydrogel-formation reaction.

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

Uniform penetration of monomers throughout the tissue is critical for 1) even polymerization throughout the tissue and 2) keeping the macro- and microstructure intact. Parts of the region of cellular structures that are not infiltrated with monomers may not be bound to the hydrogel mesh even after hybridization, and subsequent electrophoresis will result in the loss of the unbound biomolecules. Furthermore, uneven distribution of monomers may cause anisotropic expansion and reduction in volume during the electrophoretic tissue clearing and refractive index matching steps.​

If the sample contains fluorophores, cover the tube containing the sample in aluminum foil to prevent photobleaching.

If the tissues are left in the hydrogel solution for more than one day, enough protein will diffuse out of the tissue to act as a cross-inker, causing rigid gel to form around the sample. This will result in a slower rate of lipid clearing.

Stage 4 - Hydrogel tissue embedding

Steps

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.

The tubes that the tissues are transferred to should have Teflon tape applied to them before the solution is added.

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

The desiccator should have a three-way stopcock. Removal of oxygen is necessary for hydrogel-tissue hybridization because oxygen radicals may terminate the polymerization reaction.

If the caps are not unscrewed, there will be no gas exchange in the desiccator and oxygen will not be removed from the conical tubes.

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

This step is necessary to flush oxygen from all the tubing in the system.

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.

The nitrogen should not be shut off because the tubing is gas-permeable. If nitrogen flow is stopped, oxygen will diffuse back into the tubing.

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

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

It helps to have two people – one to hold the lid slightly open and another to close the tubes.

If the lids are not closed quickly enough, oxygen will re-enter the conical tubes and impede the polymerization reaction. If at this stage you find that the lids were already closed, open them slightly and repeat the de-gassing procedure.

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

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

This clearing solution with PFA must be discarded as hazardous waste according to government and institutional regulations.
Pause point: tissues may be stored in a clearing solution indefinitely following this step. If the sample contains fluorescence, be sure to cover with aluminum foil.

Stage 5 - Electrophoretic tissue-clearing

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

Steps

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

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.

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

Never start the power supply unless you have confirmed that the flow rate is satisfactory. The flow rate can be adjusted by slightly turning one of the stopcocks that surrounds the ETC chamber. A high flow rate may result in physical damage to the tissue, whereas low flow rate may result in inadequate cooling and damage the sample. Be sure to stop the voltage before stopping the pump when you shut the system off.

pH below 7 and temperatures above 37°C can result in loss of fluorescence and damage to the tissue. Be sure to check the system regularly to ensure that the temperature is not too high and that the pH has not dropped below about 7.3. If the pH is low, drain the current buffer and add new clearing solution. Lower the voltage to reduce resistive heating if the temperature is too high.

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

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

At this point, samples can be stored indefinitely at room temperature.

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.

Make sure that the container holding the sample and optical clearing solution is completely sealed and air-tight as evaporation of water from the optical clearing solution will cause the refractive index of the solution to change and will thus lower the effectiveness of optical clearing.

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

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

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

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.

Take care not to introduce any bubbles, and make sure the chamber is completely filled with optical clearing solution.

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