POL Scientific / JBM / Volume 9 / Issue 4 / DOI: 10.14440/jbm.2022.389
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Multiplexed tape-stabilized cryohistology of mineralized large animal specimens

Hannah M. Zlotnick1,2,3 Xi Jiang2 Robert L. Mauck1,2,3 Nathaniel A. Dyment1,2
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1 Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA
2 McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
3 Translational Musculoskeletal Research Center, Corporal Michael J Crescenz VA Medical Center, Philadelphia, PA 19104, USA
Published: 17 November 2022
© 2022 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

Tape-stabilized cryohistology is a powerful histological method to reinforce tissue samples during and after sectioning, enhancing the overall image quality. This technique has widely been applied to section mineralized small animal (i.e., mice, rat, rabbit) specimens, but has only been sparsely implemented for large animal samples that have a greater tendency to tear due to their increased surface area. Here, we present an optimized protocol for tape-stabilized cryohistology of undecalcified minipig vertebral body, femoral head, and temporomandibular joint samples. This protocol further develops a pipeline for sequential staining and imaging of the tape-stabilized cryosections. Images from multiple rounds of staining (endogenous bone mineral labels, aligned collagen (polarized light), tartrate resistant phosphatase (TRAP), alkaline phosphatase (AP), and toluidine blue) are overlaid to provide insight into dynamic bone remodeling. Overall, the established multiplexed tape-stabilized cryohistology protocol provides step-by-step instructions and guidance to cryosection large, mineralized tissues, and maximize data output from a single histological section.

Keywords
mineralization
bone
cryohistology
large animal
tissue interface
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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific