High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

Abstract

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward. Video LinkThe video component of this article can be found at

Amplifying Bone Marrow Progenitors Expressing α‐Smooth Muscle Actin Produce Zonal Insertion Sites During Tendon‐to‐Bone Repair

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Generation and characterization of DSPP‐Cerulean/DMP1‐Cherry reporter mice

“…Tissues were fixed overnight in 4% paraformaldehyde, decalcified and processed for cryosectioning (Dyment et al, 2016).…”

Engraftment of skeletal progenitor cells by bone-directed transplantation improves osteogenesis imperfecta murine bone phenotype

“…A subset of mice was injected with demeclocycline 2 days before they were euthanized (37.5 mg/kg, i.p., Sigma Aldrich) and bones collected and sectioned undecalcified. Cryosections (7 μm) were obtained using a Leica cryostat (Wetzler, Germany) and the tape transfer system (Section‐lab, Hiroshima, Japan) as previously described . Fluorescent images were obtained with an Axioscan slide scanner.…”