Table 1.
Comparison of different tissue clearing techniques
| Reference | Method | Principle and agent for clearing | Clearing time | Storage time | Tissue size change | IHC compatibility | Reprobing capability | Tracer compatibility | Method applied on human brain? |
|---|---|---|---|---|---|---|---|---|---|
| Spalteholz (1911) 3 | Spalteholz's technique | Ethanol and benzene to dehydrate tissue. Wintergreen oil (methyl salicylate) and benzyl benzoate (1:1) for refractive index matching | From dehydrating to clearing ~5 months | Many months | Decrease | No | No | No | Yes |
| Dodt et al. (2007) 5 | BABB/Murray's clear | Ethanol and hexane to dehydrate tissue. 1 part benzyl alcohol to 2 parts benzyl benzoate for refractive index matching |
~10 days (whole mouse brain <2 weeks old) ~1.5 days (mouse hippocampus) |
Maximum of 3 days before fluorescent signals degrade | Decrease | No | No | No | No |
| Hama et al. (2011) 9 | Scale | Immersion in ScaleA2 solution (a urea‐based aqueous clearing medium) | 2 days (sections) – 2 weeks (whole adult mouse brain) | Unknown | Increase | Limited | No | No to lipophilic dyes 11 | No |
| Ertürk et al. (2012) 8 | 3DISCO | THF to dehydrate tissue; DBE for refractive index matching | 2–5 days (whole adult mouse brain) | Days in DBE before fluorescent signals degrade (many months if AlexaFluor is used) | Decrease | Yes 24 | No |
Yes to CTb 24
No to lipophilic dyes |
No |
| Kuwajima et al. (2013) 11 | ClearT | Immersion in graded formamide to 95% formamide | ~2.5 days (whole mouse brain); 35 mins (1 mm sections) | Formamide is unsuitable for long‐term storage | No change or mild expansion | No | No | Yes | No |
| Kuwajima et al. (2013) 11 | ClearT2 | Immersion in graded formamide to 50% formamide/20% polyethylene glycol (PEG) solution | ~18 h (whole mouse brain); up to 75 mins (1 mm sections) | Formamide is unsuitable for long‐term storage | No change or mild expansion | Yes | No | Yes | No |
| Chung et al. (2013) 13 | CLARITY | Formation of hydrogel‐tissue hybrid matrix with acrylamide and bisacrylamide. Clearing of tissue with 4% SDS in boric acid buffer | ~2 weeks (whole adult mouse brain) | Many months | Transient increase | Yes | Yes | No to lipophilic dyes 25 | Yes |
| Ke et al. (2013) 10 | SeeDB | Immersion in graded fructose to saturated fructose with 0.5% α‐thioglycerol | About 3 days (embryonic mouse brain) | Up to 1 week in SeeDB solution; longer in PBS | No change | No | No | Yes | No |
| Susaki et al. (2014) 12 | CUBIC | Immersion in N,N,N′,N′‐tetrakis(2‐hydroxypropyl)ethylenediamine (25%), Triton X‐100 (wt 15%) and urea (~4 M) for tissue clearing. Then immersion in sucrose (50%), 2,20,20′‐nitrilotriethanol (10%), Triton X‐100 (v/v 0.1%) and urea (~4 M) for refractive index matching | 2 weeks (whole mouse brain) | Many months immersed in 20% (w/v) sucrose in PBS, and stored in OCT compound at −80°C | Transient increase | Yes | No | Unknown | No |
BABB, benzyl alcohol–benzyl benzoate; CTb, cholera toxin B subunit; DBE, dibenzyl ether; OCT, optimal cutting temperature; PBS, phosphate‐buffered saline; SDS, Sodium dodecyl sulphate; THF, tetrahydrofuran.