Advances in CLARITY‑based tissue clearing and imaging (Review)

Abstract

CLARITY is a novel tissue clearing technique that transforms intact biological tissues into a nanoporous hydrogel-tissue hybrid, preserving anatomical structures, proteins and nucleic acids. The hydrogel-based structure is transparent after the removal of lipids and permits several rounds of immunostaining and imaging. This technique provides an ideal way for researchers to examine the central nervous system (i.e., mouse brain and spinal cord) intact. CLARITY was selected as one of ten breakthroughs in 2013 by Science. However, the original CLARITY technique still has severe technical limitations which impede its application in wider fields. Therefore, many modified clearing methods based on CLARITY have emerged. As all CLARITY-based tissue clearing techniques involve similar procedures, the present review attempted to divide these methods into individual procedures in order to provide new ways to test and combine tissue clearing methods. Furthermore, the combination of clearing methods could help to determine the optimal method for clearing and imaging large samples.

The Dynamic Relationship of Breast Cancer Cells and Fibroblasts in Fibronectin Accumulation at Primary and Metastatic Tumor Sites

“…After fixation in 10% formalin, one lobe of each lung and half of each primary tumor underwent tissue clearing following the X-CLARITY Systems protocol (Logos Biosystems, Annandale, VA, USA) [55]. Briefly, samples were submerged for 24 h at 4 • C in hydrogel initiator solution (1 part 25% (w/v) X-CLARITY Polymerization Initiator with 100 parts X-CLARITY Hydrogel Solution) (Cat#C1310X).…”

“…Cleared samples were immunolabeled using the c-PRESTO (centrifugal pressure related efficient and stable transfer of macromolecules into organs) technique [55,56]. Briefly, samples were blocked with 6% bovine serum albumin and 0.1% TritonX-100 for 24 h and stained with FN (Sigma, Cat#3648) for 2 h at 4000 rpm and DAPI.…”

StereoMate: 3D Stereological Automated Analysis of Biological Structures

“…Combining these methods with standard immunohistochemical approaches, however, has proved challenging. The process of tissue clearing appears to have severe consequences for tissue antigenicity, resulting in suboptimal antibody labelling 10 . With the current best approach for cleared tissue immunohistochemistry 4,8 , it is recommended to check whether antigenciity is sufficiently well maintained following the initial lipid elution with different antigens.…”

Toward Better Medical Diagnosis: Tissue Optical Clearing

“…Chemical optical clearing techniques are sometimes termed "tissue transformation" because it depends on removing the lipid contents from the tissue sample in order to reduce the refractive index mismatching through the tissue [16]. Lipid extraction is implemented using electrophoretic tissue clearing (ETC) chamber to apply an electric field [17] or can be achieved passively without ETC [18].…”