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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.
|Beuthanasia-D||Schering-Plough Animal Health Corp.|
|32% Paraformaldehyde (PFA)||Electron Microscopy Sciences, 15714-S|
|40% Acrylamide solution||Bio-Rad, 161-0140|
|10X PBS||Invitrogen, 70011-044|
|Ultrapure distilled water||Invitrogen, 10977-015|
|Boric acid||Sigma-Aldrich, B7901|
|Sodium dodecyl sulfate (SDS)||Sigma-Aldrich, L3771|
|Lithium hydroxide monohydrate||Sigma-Aldrich, 254274|
|Diatrizoic acid||Sigma-Aldrich, D9268|
|60% Iodixanol||Sigma-Aldrich, D1556|
|Triton-X (TX)||Sigma-Aldrich, T8787|
|Sodium azide||Sigma-Aldrich, S2002|
|1X PBS||Invitrogen, 10010-023|
|Transcardial perfusion of fixatives and hydrogel monomers|
|Dissection board (Styrofoam lid is fine)||–|
|20 ml syringes with luer lock ends||Fisher Scientific, 14-820-19|
|1 ml syringes||Terumo, SS-01T|
|Winged infusion sets||Terumo, SV-25BLK|
|Needles||Fisher Scientific, BD 305109|
|Absorbent pads||VWR, 56616-032|
|50 mL Falcon tubes||BD Falcon, 352070|
|Guillotine, for sacrificing larger animals||Kent Scientific, DCAP|
|Surgical scissors||Fine Science Tools, 14130-17|
|Fine scissors||Fine Science Tools, 14137-10|
|Hemostats||Fine Science Tools, 13011-12|
|Forceps||Fine Science Tools, 11050-10, 11251-10|
|Spatula||Fine Science Tools, 10092-12|
|Desiccator with 3-way stopcock||VWR, 24988-197|
|Vacuum pump||Buchi, 071000|
|Compressed nitrogen tank||AirGas, NI UHP300|
|Compressed gas tank pressure regulator||AirGas, Y11215B580|
|Teflon tape||McMaster-Carr, 4591K11|
|3/8” tubing||McMaster-Carr, 5155T36|
|3/8” to 1/4" barbed tubing connector||McMaster-Carr, 5463K633|
|ETC clearing system|
|Buffer Filter with Light-Blocking Blue Bowl||McMaster-Carr, 4448K35|
|Platinum wire with 0.5mm diameter||Sigma, 267201|
|Bottle for Chamber fabrication||Nalgene via Amazon, 2118-0002|
|Nalgene Straight Side Jar – Poly, 32oz||Nalgene via Amazon|
|Single barbed tube fitting (7/16” hex for 1/4” tubing)||McMaster-Carr, 5463K245|
|Tube to tube coupling for 3/32” to 1/16” tubing||McMaster-Carr, 5117K51|
|3M Duo adhesive dispenser||McMaster-Carr, 7467A43|
|3M Duo adhesive-mixing applicators||McMaster-Carr, 7467A12|
|3M Duo adhesive cartridges||McMaster-Carr, 746A17|
|Sample holder||BD Falcon, 352340|
|Bio-Rad HC PowerPac System||Bio-Rad, 164-5052|
|Banana to Large Alligator Test Lead Set||Elenco, TL16|
|Clear 1/4" tubing||McMaster, 5155T26|
|Clear 5/8” tubing||McMaster, 5155T46|
|1/4" wye connector||McMaster, 53055K155|
|4x Chemical resistant stopcock 1/4" to 1/4"||McMaster, 48285K24|
|5/8" to 1/4" tubing connection||McMaster-Carr, 2974K271|
|Elbow connection 1/4" male pipe to 1/4" barbed fitting||McMaster-Carr, 5463K489|
|Elbow connection 1/4" barbed fitting||McMaster-Carr, 5463K594|
|Rubber grounding plug||Leviton via Amazon, L00-515PR-000|
|Magnetic water pump||Pan World via Premium Aquatics, NH-10PX|
|KWIK-SIL||World Precision Instruments, KWIK-SIL|
|Willco-dish||Ted Pella, 14032-120|
|Blu-tack reusable adhesive||Blu-Tack via Amazon|
In principle, any tissue type from any animals of any age with or without fluorescence can be used. In the previous paper1, we demonstrated that CLARITY is compatible with 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 can be directly imaged; tissues without fluorescent proteins can be labelled with antibodies or RNA probes1 for subsequent imaging.
Hydrogel monomer solution
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:
|UltraPure water||26 mL|
|40% Acrylamide solution||4 mL|
|Initiator solution||1 mL|
|10X PBS||4 mL|
|32% PFA||5 mL|
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.
CAUTION: Make sure to keep all reagents and the final solution on ice at all times. The hydrogel polymerization reaction is triggered by heat.
SDS clearing solution
Adjust the following solution to pH 8.5 using boric acid.
|Lithium hydroxide monohydrate||20 mM|
Make a solution consisting of 0.1% Triton-X and 0.1% Sodium Azide using 1X PBS.
|1X PBS||500 mL|
|Sodium azide||500 mg|
Optical clearing solution (PROTOS)
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. Use a stir bar (or shake if necessary) to fully dissolve the powders at each step. Do not use heat when mixing the solution, as this will cause a color change.
This solution should be stored carefully to ensure that no water is lost, as just a small amount of evaporation will result in precipitation. Teflon tape can be used to increase the security of the bottle’s seal, and 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. The optical clearing solution in the case would look as follows:
|47% Iodixanol solution in water*||10 mL|
|Diatrizoic acid||4.24 g|
*(To create this, add approximately 2.75 mL water to every 10 mL of 60% iodixanol solution)
Note: reagents should be added in order
Perfusion and Tissue Preparation
21) Carefully place the sample in between the Blu-Tack pieces and add about 20 μL of optical clearing solution to the sample.
22) 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.
23) 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.
24) Cover this construction with aluminum foil and store it away safely to cure. After about 20 minutes, the sample is ready for imaging.