Methods for the visualization and analysis of extracellular matrix protein structure and degradation

Annemarie K Leonard; Elizabeth A Loughran; Yuliya Klymenko; Yueying Liu; Oleg Kim; Marwa Asem; Kevin McAbee; Matthew J Ravosa; M Sharon Stack

doi:10.1016/bs.mcb.2017.08.005

Methods for the visualization and analysis of extracellular matrix protein structure and degradation

Methods Cell Biol. 2018:143:79-95. doi: 10.1016/bs.mcb.2017.08.005.

Authors

Annemarie K Leonard¹, Elizabeth A Loughran², Yuliya Klymenko¹, Yueying Liu¹, Oleg Kim³, Marwa Asem², Kevin McAbee¹, Matthew J Ravosa¹, M Sharon Stack⁴

Affiliations

¹ University of Notre Dame, Notre Dame, IN, United States; Harper Cancer Research Institute, University of Notre Dame, South Bend, IN, United States.
² University of Notre Dame, Notre Dame, IN, United States; Harper Cancer Research Institute, University of Notre Dame, South Bend, IN, United States; Integrated Biomedical Sciences Graduate Program, University of Notre Dame, Notre Dame, IN, United States.
³ University of Notre Dame, Notre Dame, IN, United States; Harper Cancer Research Institute, University of Notre Dame, South Bend, IN, United States; University of California, Riverside, Riverside, CA, United States.
⁴ University of Notre Dame, Notre Dame, IN, United States; Harper Cancer Research Institute, University of Notre Dame, South Bend, IN, United States. Electronic address: sstack@nd.edu.

Abstract

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders.

Keywords: Collagen; Extracellular matrix; Histology; Immunohistochemistry; Invasion; SDS-PAGE; Scanning electron microscopy; Second harmonic generation microscopy; Trichrome staining; Zymography.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Collagen Type I / chemistry
Collagen Type I / ultrastructure*
Connective Tissue Diseases / etiology
Connective Tissue Diseases / pathology
Extracellular Matrix / chemistry
Extracellular Matrix / ultrastructure*
Humans
Immunohistochemistry / methods
Microscopy, Electron, Scanning / instrumentation
Microscopy, Electron, Scanning / methods
Molecular Imaging / instrumentation
Molecular Imaging / methods*
Proteolysis*
Staining and Labeling / instrumentation
Staining and Labeling / methods*

Substances

Collagen Type I

Abstract

Publication types

MeSH terms

Substances

Grants and funding