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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.

Summary The mononuclear phagocytic system (MPS), comprised of monocytes, macrophages, and dendritic cells, is essential in tissue homeostasis and in determining the balance of the immune response through its role in antigen presentation. It has been identified as a therapeutic target in infectious disease, cancer, autoimmune disease and transplant rejection. Here, we review the current understanding of the immunophenotype and function of the MPS in normal human liver. Using well-defined selection criteria, a search of MEDLINE and EMBASE databases identified 76 appropriate studies. The majority (n = 67) described Kupffer cells (KCs), although the definition of KC differs between sources, and little data were available regarding their function. Only 10 papers looked at liver dendritic cells (DCs), and largely confirmed the presence of the major dendritic cell subsets identified in human blood. Monocytes were thoroughly characterized in four studies that utilized flow cytometry and fluorescent microscopy and highlighted their prominent role in liver homeostasis and displayed subtle differences from circulating monocytes. There was some limited evidence that liver DCs are tolerogenic but neither liver dendritic cell subsets nor macrophages have been thoroughly characterized, using either multi-colour flow cytometry or multi-parameter fluorescence microscopy. The lobular distribution of different subsets of liver MPS cells was also poorly described, and the ability to distinguish between passenger leukocytes and tissue resident cells remains limited. It was apparent that further research, using modern immunological techniques, is now required to accurately characterize the cells of the MPS in human liver. [ABSTRACT FROM AUTHOR]