Table 4 A comparison of several methods for generating optically clear tissues for immunofluorescence staining

Thick sections (100–150 µm)^{56,57,58,59,60}

Bone marrow, spleen, muscle, heart, gut, skin, lung, etc.

Yes

Yes

Not applicable

Simple to implement; inexpensive; reduces background signals; non-toxic

Spatial information is limited; difficult to study rare populations

BABB^{4,29,67,71,76,88}

Bone marrow, spleen, muscle, heart, gut, skin, lung, etc.

Yes

Yes

Not applicable

Simple to implement; inexpensive; reduces background signals; non-toxic

Spatial information is limited; difficult to study rare populations

PEGASOS⁷⁷

Bones, brain, muscles, heart, lung, kidney, liver, pancreas, spleen, muscle, stomach, intestine, skin

Yes

Yes

~12–14 days for hard tissues, ~7 days for soft tissues

Strong clearing capability for both soft and hard tissues; no need to immunostain endogenous fluorescent proteins

Soft tissues undergo variable shrinkage; complicated protocol

Eci^65,89,90,91

Kidney, heart, calvarial and long bones

Yes

No

~3 days

Non-toxic clearing reagent

Difficult long-term storage

iDISCO^{38,66,70,74,78}

Brain, heart, vertebral arteries, lung, liver, prostate

Yes

No fluorescence is quenched

>3 h for small organs, 1–2 weeks for the whole mouse embryo and complex adult organs

Strong clearing capability for soft tissues

Toxic and thus careful handling is required; tissues are fragile; not all epitopes are applicable; soft tissues undergo shrinkage

Bone CLARITY⁷⁵

Bone

Yes

Yes

28 days

Imaging depth of up to 1.5 mm for the epiphysis of long bones; integrity of the bone marrow is retained

Poor penetration of relatively large antibodies