Sample Preparation of Optically Cleared Liver Tissue to Identify Liver Macrophages Using 3D Microscopy

Abstract

Histology, or the study of tissue microanatomy, is essential to understanding the in situ function of varying cell types within an organ. How cells are distributed throughout organs provides an indication of how they interact with other cells and structures within the organ microanatomy. The tortuous shape and large size of liver macrophages limits the value of standard tissue thickness of 5–10 μm. As a result, imaging of specimens ideally thicker than 100 μm is necessary to investigate the liver microanatomy and the how macrophages are distributed within this. Modern methods of microscopy, such as confocal and light sheet microscopy, allow for the analysis of tissue specimens of a thickness well beyond 100 μm in the z-dimension. Liver tissue is an opaque tissue, and as a result, different techniques are needed to ameliorate light diffraction within the tissue. These techniques, in conjunction with antibody staining and refractive index matching of the tissue, have allowed researchers to image liver tissue specimens of more than 100 μm thickness. Two of these techniques are modified versions of the clearing methods known as clearing-enhanced 3D (Ce3D) and fructose, urea, and glycerol for imaging (FUnGI). Here, we discuss the steps involved in preparing tissue specimens for optically clearing tissue using Ce3D and FUnGI for subsequent analysis of the distribution of macrophages in three dimensions using a confocal microscope.