Abstract
Alterations in the proteome of a tissue in different settings, as assessed by difference gel electrophoresis, can be verified for single proteins using immunohistochemistry. In fluorescence immunohistochemistry, an antibody to a particular antigen is applied to tissue sections, and fluorophores conjugated to a secondary antibody allow for the detection of target antigen with fluorescent microscopy. Visual comparison is sufficient for the detection of significant alterations in the abundance of a certain protein in different settings. Additionally, unlike large-scale proteome analyses and Western blot methods, expression of target protein can be analyzed at the cellular level by immunohistochemistry. In this chapter, a protocol for the application of fluorescence immunohistochemistry for the detection of dystrophin in skeletal muscle sections is outlined, including sample preparation, tissue sectioning, and immunostaining.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Coons AH, Kaplan MH (1950) Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med 91:1–13
Murphy S, Henry M, Meleady P et al (2015) Simultaneous pathoproteomic evaluation of the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of Duchenne muscular dystrophy. Biology (Basel) 4:397–423
Murphy S, Dowling P, Zweyer M et al (2016) Proteomic analysis of dystrophin deficiency and associated changes in the aged mdx-4cv heart model of dystrophinopathy-related cardiomyopathy. J Proteome 11(145):24–36
Matsumura K, Campbell KP (1994) Dystrophin-glycoprotein complex: its role in the molecular pathogenesis of muscular dystrophies. Muscle Nerve 17:2–15
Allen DG, Gervasio OL, Yeung EW et al (2010) Calcium and the damage pathways in muscular dystrophy. Can J Physiol Pharmacol 88:83–91
Mallouk N, Jacquemond V, Allard B (2000) Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels. Proc Natl Acad Sci U S A 97:4950–4955
Kumamoto T, Fujimoto S, Ito T et al (2000) Proteasome expression in the skeletal muscles of patients with muscular dystrophy. Acta Neuropathol 100:595–602
Menke A, Jockusch H (1991) Decreased osmotic stability of dystrophin-less muscle cells from the mdx mouse. Nature 349:69–71
Holland A, Carberry S, Ohlendieck K (2013) Proteomics of the dystrophin-glycoprotein complex and dystrophinopathy. Curr Protein Pept Sci 14:680–697
Carberry S, Zweyer M, Swandulla D et al (2013) Application of fluorescence two-dimensional difference in-gel electrophoresis as a proteomic biomarker discovery tool in muscular dystrophy research. Biology (Basel) 2:1438–1464
Carberry S, Zweyer M, Swandulla D et al (2012) Proteomics reveals drastic increase of extracellular matrix proteins collagen and dermatopontin in the aged mdx diaphragm model of Duchenne muscular dystrophy. Int J Mol Med 30:229–234
Mundegar RR, Franke E, Schäfer et al (2008) Reduction of high background staining by heating unfixed mouse skeletal muscle tissue sections allows for detection of thermostable antigens with murine monoclonal antibodies. J Histochem Cytochem 56:969–975
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Mundegar, R.R., Zweyer, M., Swandulla, D. (2018). Immunofluorescence Microscopy for DIGE-Based Proteomics. In: Ohlendieck, K. (eds) Difference Gel Electrophoresis. Methods in Molecular Biology, vol 1664. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7268-5_23
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7268-5_23
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7267-8
Online ISBN: 978-1-4939-7268-5
eBook Packages: Springer Protocols