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Nat Protoc. 2019 Dec;14(12):3395-3425. doi: 10.1038/s41596-019-0225-8. Epub 2019 Nov 8.

Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

Author information

1
Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), Copenhagen, Denmark. alejandro.mayorca@bric.ku.dk.
2
Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), Copenhagen, Denmark.
3
Department of Laboratory Medicine, Division of Translational Cancer Research, Lund University, Lund, Sweden.
4
Center for Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany.
5
Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany.
6
Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
7
Department of Pediatrics and Adolescent Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany.
8
Institute for Dental Research and Oral Musculoskeletal Biology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany.
9
Dr. Rolf M. Schwiete Research Unit for Osteoarthritis, Orthopedic University Hospital Friedrichsheim gGmbH, Frankfurt, Germany.
10
Department of Biochemistry II, Faculty of Medicine, Oita University, Oita, Japan.
11
Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), Copenhagen, Denmark. janine.erler@bric.ku.dk.
12
Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), Copenhagen, Denmark. raphael.reuten@bric.ku.dk.

Abstract

The extracellular matrix (ECM) is a major regulator of homeostasis and disease, yet the 3D structure of the ECM remains poorly understood because of limitations in ECM visualization. We recently developed an ECM-specialized method termed in situ decellularization of tissues (ISDoT) to isolate native 3D ECM scaffolds from whole organs in which ECM structure and composition are preserved. Here, we present detailed surgical instructions to facilitate decellularization of 33 different mouse tissues and details of validated antibodies that enable the visualization of 35 mouse ECM proteins. Through mapping of these ECM proteins, the structure of the ECM can be determined and tissue structures visualized in detail. In this study, perfusion decellularization is presented for bones, skeletal muscle, tongue, salivary glands, stomach, duodenum, jejunum/ileum, large intestines, mesentery, liver, gallbladder, pancreas, trachea, bronchi, lungs, kidneys, urinary bladder, ovaries, uterine horn, cervix, adrenal gland, heart, arteries, veins, capillaries, lymph nodes, spleen, peripheral nerves, eye, outer ear, mammary glands, skin, and subcutaneous tissue. Decellularization, immunostaining, and imaging take 4-5 d.

PMID:
31705125
DOI:
10.1038/s41596-019-0225-8

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