Simple and Rapid Tissue Clearing Method for Three‐Dimensional Histology of the Pancreas

Abstract

Previously, high-resolution three-dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide-based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel-embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA). The whole procedure is less time-consuming and less error-prone, and can be completed within a week, compared to conventional CLARITY protocols that may take weeks to complete. Here, the detailed stepwise procedures involved in the simplified CLARITY workflow are applied to the pancreas of wild-type and gene-knockout 6-week old mice expressing green fluorescent protein (GFP) under the mouse insulin 1 promoter (MIP-GFP). This technique could facilitate high-resolution, three-dimensional imaging of pancreatic islets and comparison between different mouse genotypes under different disease and treatment conditions. © 2017 by John Wiley & Sons, Inc.

A Light sheet fluorescence microscopy and machine learning-based approach to investigate drug and biomarker distribution in whole organs and tumors

“…Innovation in tissue clearing and LSFM methods has been instrumental in helping close a key gap in imaging technology – enabling the acquisition of three-dimensional spatial information for whole organs and tumors, without compromising on subcellular resolution and multiplexing. Applications for this burgeoning technology have rapidly spanned spatial assessment of a variety of different organs such as the pancreas 29, 30 , heart 31 , kidneys 32 , brain 33 , liver 34 , bone marrow 35 , and even whole organisms 36, 37 . Here we explored oncology and neuroscience - two therapeutic areas that could significantly benefit from tissue clearing and LSFM applications to help drive drug discovery.…”

Current Status of Tissue Clearing and the Path Forward in Neuroscience

“…Immunolabeling, including IHC and IF, is the standard method to visualize antigens, which circum- vents species restriction and allows shift spectra 120 and signal amplification. 121 When combined with tissue clearing, immunostaining demonstrates considerable compatibility, with a few exceptions, such as protocols omitting the delipidation step, which renders tissue poorly accessible to antibodies (e.g., SeeDB 58 and FRUIT 19 ). However, issues with staining quality have been reported, such as uneven staining and short-term fluorescence preservation, as well as complaints about the staining efficiency (e.g., low throughput, high cost, and time-consuming).…”

“…Immunolabeling, including IHC and IF, is the standard method to visualize antigens, which circumvents species restriction and allows shift spectra and signal amplification . When combined with tissue clearing, immunostaining demonstrates considerable compatibility, with a few exceptions, such as protocols omitting the delipidation step, which renders tissue poorly accessible to antibodies (e.g., SeeDB and FRUIT).…”