Ce3D™ Tissue Clearing

Biological tissues are generally composed of proteins, lipids, and water, each of which has a different refractive index (RI). Differences in refractive indices, or RI mismatch, cause the scattering of light in the tissue and result in tissue opacity. Tissue clearing reagents aim to reduce light scattering by normalizing the RI throughout the tissue, thus making the tissue transparent. Optical clearing is a valuable application that allows us gain a better understanding of spatial composition, phenotypic and subtype identity, and cellular networks. Our Ce3D™ Tissue Clearing Solution pairs perfectly with our expansive portfolio of antibodies, generating mesmerizing 3D images with unparalleled depth.

The Ce3D™ tissue clearing system was developed by the Laboratory of Immune System Biology (LISB) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH). The original methods can be reviewed below:

Watch our video explaining the capabilities of Ce3D™.

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3D IHC Data

3D Imaging

The images above are representative 3D IHC images of 500 μm-thick mouse samples. The sections were optically cleared after immunostaining, mounted in sample chambers, and imaged with a 10X or 20X objective.

From left to right:

Mouse intestine: CD45 (green), CD326 (blue), Tubulin β (magenta)
Mouse spleen: CD3ε (magenta), CD169 (green), I-A/I-E (blue)
Mouse intestine: CD45 (magenta), Tubulin β (green)
Mouse liver: DAPI (blue), CLEC4F (magenta), CD324 (yellow)

Watch dozens of our 3D imaging videos.

Optical Clearing Using Ce3D™ Tissue Clearing Solution

Representative 500 µm-thick mouse tissue sections before and after tissue clearing. Mice were perfused and organs were fixed with 1% (A) or 4% (B) PFA. Clearing was reversed by immersing tissues in PBS for one hour. 1% PFA is recommended for best clearing effect. 4% PFA is recommended for optimal epitope preservation and immunostaining.

Optical Clearing Using Ce3D™ Tissue Clearing Solution

What is Ce3D™?

Ce3D™ (clearing-enhanced 3D) Tissue Clearing Reagent is a hydrophilic and RI-matching solution, making it easy-to-use for the rapid and robust clearing of diverse tissues. Ce3D™ has an RI of ~1.5 and is compatible with multicolor fluorescence microscopy. The Ce3D™ tissue clearing technique works by simple immersion of tissue into the clearing reagent, and does not require special equipment or containers. Ce3D™ offers many advantages:

Key Features

Simple, one-step passive clearing process
Preservation of fluorescent signal, tissue architecture, and cell morphology
Reversible to allow 2D IHC and other downstream applications
Available as pre-assembled kits or as individual reagents, saving time on buffer preparation
Validated with over 60 BioLegend antibody clones and conjugated formats

Ce3D™ Tissue Clearing Kits

Test Size	Cat. No.	Ce3D™ Tissue Clearing Solution	Ce3D™ Permeabilization/Blocking Buffer	Ce3D™ Antibody Diluent Buffer	Ce3D™ Wash Buffer
10 Tests	427701	5 mL	15 mL	15 mL	50 mL
50 Tests	427702	25 mL	50 mL	50 mL	250 mL

Ce3D™ Tissue Clearing Individual Reagents

Reagent	Size (µL)	Cat. No.
Ce3D™ Tissue Clearing Solution	5 mL \| 25 mL	427703 \| 427704
Ce3D™ Permeabilization/Blocking Buffer	25 mL \| 50 mL	427705 \| 427706
Ce3D™ Antibody Diluent Buffer	15 mL \| 50 mL	427707 \| 427708
Ce3D™ Wash Buffer	50 mL \| 250 mL	427709 \| 427710

Ce3D™ Tissue Clearing Kit Staining Protocol for Microscopy

View our step-by-step protocol video
for Ce3D™ reagents.

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Our protocol and troubleshooting guide provides an in-depth optimized protocol, complete with notes and commentary for each step. The guide also outlines the entire procedure, leading you through perfusion, fixation, sectioning, staining, tissue clearing, and mounting steps. The process itself can typically be completed within 7 days.

In addition, the guide contains suggestions for resolving potential issues you may encounter as you optimize your experiment. Read through it for tips on fixation, uneven staining, and high background. For additional information or help, contact our Technical Services team.

Recommended Clearing Time for Different Tissues

Time	Tissue
2 hours	Intestine, lymph node
4 hours	Spleen, thymus, lung
> 8 hours	Kidney, liver, testis, brain