Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Summary

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Abstract

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel’s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

Introduction

The musculoskeletal system is a multicomponent system composed of muscle, connective tissue, tendon, ligament, cartilage, and bone, innervated by nerves and vascularized by blood vessels¹. The skeletal tissues develop with increasing cellular heterogeneity and structural complexity. Cartilage and bone develop from the same osteochondroprogenitor lineage and are highly related. Embryonic cartilage and bone develop in association with muscles, nerves, blood vessels, and undifferentiated mesenchyme. Cartilage may also be surrounded by bone, such as Meckel's cartilage and condylar cartilage within the mandibular bone. These tissues are anatomically....

Protocol

Tissues from mice were obtained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and study protocols were approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai.

1. Preparation of Fresh Frozen Specimen

1. Dissect the embryo or tissue of interest. Embed the sample in a disposable embedding mold with optimal cutting temperature (OCT) compound. Adjust the orientation of the specimen with a tip or needle.
2. Rapidly freeze the samples in a dry ice/methyl-2-butane bath. Continue to the next step or store at -80 °C.
   ....

Results

Coronal sections of fresh frozen mouse tissues at E16.5 were used to demonstrate the isolation and collection of Meckel's cartilage (MC), condylar cartilage, and mandibular bone by LCM. Mouse embryos at E16.5 were dissected and embedded in cryogenic molds with OCT compound. Samples in molds were rapidly frozen in a dry ice and methyl-2-butane bath and stored at -80 °C.

To demonstrate cresyl violet staining of cartilage and bone, cryosectioning in the coronal plane was performed and samples wer.......

Discussion

LCM enables the isolation of enriched or homogenous cell populations from heterogeneous tissues. Its advantages include rapid and precise capture of cells in their in vivo context, while potential disadvantages include it being time consuming, expensive, and limited by the need for the user to recognize distinct subpopulations within a specified sample³⁰. This protocol provides details of LCM of mouse embryonic cartilage and bone, highlighting the use of cresyl violet staining in a rapid .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Methylbutane	ThermoFisher Scientific	O3551-4
Bioanalyzer	Agilent	G2939BA
Centrifuge tube	ThermoFisher Scientific	339653	Conical sterile polypropylene centrifuge tubes, 50 mL
Cresyl violet acetate	Sigma-Aldrich	C5042
Cryostat	Leica Biosystems	CM3050 S
Delicate task wiper	ThermoFisher Scientific	06-666
Disposable embedding mold	ThermoFisher Scientific	1220
Distilled water	Invitrogen	10977-015	DNase/RNase-Free
Ethanol, absolute (200 proof)	ThermoFisher Scientific	BP2818	Molecular biology grade
Glass PEN membrane slide	Leica Microsystems	11505158
LCM system	Leica Microsystems	Leica LMD6500
Microscope cover glass	ThermoFisher Scientific	12-545FP
Microscope slides	ThermoFisher Scientific	12-550-15
OCT compound	Electron Microscopy Sciences	102094-106
PCR tube with flat cap, 0.5 mL	Axygen	PCR-05-C	LCM collection tubes
Permanent mounting medium	Vector Laboratories	H-5000
RNA isolation kit	ThermoFisher Scientific	KIT0204
RNase decontamination agent	Sigma-Aldrich	R2020	RNase decontamination agent for cleaning surfaces
Xylene	Sigma-Aldrich	214736