Three-dimensional histochemistry and imaging of human gingiva

Abstract

In the present study, 3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network. Fifteen human gingiva samples without signs of inflammation were cleared using a mixture of 2-parts benzyl benzoate and 1-part benzyl alcohol (BABB), after being immunofluorescently stained for CD31, marker of endothelial cells to visualize blood vessels in combination with fluorescent DNA dyes. Samples were imaged in 3D with the use of confocal microscopy and light-sheet microscopy and image processing. BABB clearing caused limited tissue shrinkage 13 ± 7% as surface area and 24 ± 1% as volume. Fluorescence remained intact in BABB-cleared gingiva samples and light-sheet microscopy was an excellent tool to image gingivae whereas confocal microscopy was not. Histochemistry on cryostat sections of gingiva samples after 3D imaging validated structures visualized in 3D. Three-dimensional images showed the vascular network in the stroma of gingiva with one capillary loop in each stromal papilla invading into the epithelium. The capillary loops were tortuous with structural irregularities that were not apparent in 2D images. It is concluded that 3D histochemistry and imaging methodology described here is a promising novel approach to study structural aspects of human gingiva in health and disease.

Poor perfusion of the microvasculature in peritoneal metastases of ovarian cancer

“…Sections were encircled with a PAP pen (Dako, Glostrup, Denmark) and incubated using TBS containing 3% normal goat serum (Dako) and 0.1% Triton-X for 1 h to further reduce non-specific background staining and for permeabilization of the sections. Sections were subsequently incubated overnight at 4 °C with primary antibodies against paired-box gene 8 (PAX-8 363M-15; clone MRQ50, dilution 1:50; Cellmarque, Rocklin, CA, USA), which specifically stains EOC cells, VEGF (sc-152; Santa Cruz, Biotechnology, Dallas, TX, USA), cluster of differentiation 31 (CD31; M0823, clone JC70A, dilution 1:250, Dako), which specifically stains endothelial cells [26][27][28][29]. Next, sections were incubated with 3,3′-diaminobenzidine (Dako) for 10 min, followed by one washing step with tap water to stop the peroxidase enzyme reaction.…”

“…Samples were left overnight in 100% methanol and the next day incubated for 3 h in 66% DCM/33% methanol at RT, 2 × in 100% DCM (Sigma) for 15 min and finally incubated in dibenzyl ether (Sigma) (no shaking) until samples were transparent. Upon clearing, samples were immediately imaged with light sheet fluorescence microscopy using the Ultramicroscope (LaVision BioTec, Bielefeld, Germany) [28]. Inspector Software (LaVision, BioTec) was used for settings adjustment and image acquisition.…”

“…Inspector Software (LaVision, BioTec) was used for settings adjustment and image acquisition. Image analysis was performed using Imaris 9.2 or higher [28].…”

Advances in Ex Situ Tissue Optical Clearing

“…The suitability of dehydration methods for other hard, connective tissue, gingiva, accompanied by successful anti‐CD31 staining, was also reported by Azaripour et al . A group led by Gradinaru has recently contributed to the development of bone‐specific CLARITY protocol.…”

Through-focus or volumetric type of optical imaging methods: a review

“…Because of the opacity of such tissues, threedimensional imaging is typically limited to a depth of 500 μm to 1000 μm (72, 73). However, in some studies LSM appears to outperform confocal microscopy (10). …”