Three‐dimensional Histochemistry and Imaging of Extracellular Matrix‐rich Human Tissues

For 3‐dimensional (3D) imaging of tissues, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1. clearing of the opaque tissue to render it transparent for microscopy, 2. fluorescence labeling of the tissues and 3. 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most of these clearing techniques are based on dissolving membranes and have been applied to the central nervous system and other tissues that contain relatively low amounts of extracellular matrix (ECM). Tissues containing large amounts of ECM such as dermis in skin and gingiva are difficult to clear in this way. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether (DBE) are the most successful methods for clearing ECM‐rich tissues. The principle of this type of clearing is that the entire tissue has one refractive index, that of BABB or DBE. We applied the BABB method in combination with immunofluorescence staining of CD31, an endothelial marker, and YOYO‐3, a fluorescent DNA dye, light‐sheet microscopy and Imaris‐based image analysis and processing to investigate the vasculature network in 3D in 12 human gingiva samples without signs of inflammation. 3D images showed the vascular network in 3D in the lamina propria of gingiva with loops into the papillae. The vascular network showed a high degree of tortuosity and irregularities in structure which are not apparent in images of sections of gingiva. This 3D histochemistry and imaging methodology is an excellent tool to study structural aspects of human gingiva that are involved in diseased periodontium. Support or Funding Information No funding.