Abstract
The study of brain pathology by fluorescence microscopy finds in the autofluorescence of the tissue an additional difficulty for the recognition of markers of interest. In particular, in the immunofluorescence study of brains from Alzheimer’s disease (AD) patients, several approaches have been attempted to eliminate or mask the presence of autofluorescent aggregates. In the present work, we propose a method to characterize by fluorescent microscopy senile plaques discriminating them from autofluorescent aggregates, such as lipofuscin granules.
This work describes four protocols carried out in human brain tissue of patients with AD, covering adequate tissue preparation, immunofluorescence acquisition, and data analysis:
-
1.
Tissues processing of frozen samples for optimal epitope conservation.
-
2.
Analysis of the fluorescence emission spectrum of the tissue by performing a confocal microscopy λ-scan.
-
3.
Analysis of fluorescence emission of both intact and formic acid-treated tissues in four channels corresponding to the emission in blue, green, near red, and far-red regions.
-
4.
Analysis a specific immunostaining of amyloid beta in senile plaques, using fluorescent-labeled antibodies by using specific emission channels to avoid detection of tissue autofluorescence.
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
Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York. ISBN: 978-0-387-31278-1
Naresh K (2014) Applications of fluorescence spectroscopy. J Chem Pharm Sci 5(Special Issue):18–21. ISSN: 0974-2115
An WF (2009) Fluorescence-based assays. Methods Mol Biol 486:97–107
Monice M (2005) Cell and tissue autofluorescence research and diagnostic applications. Biotechnol Annu Rev 11:227–256
Firląg M, Kamaszewski M, Gaca K et al (2013) Age-related changes in the central nervous system in selected domestic mammals and primates. Postepy Hig Med Dosw (Online) 67:269–275
Grune T, Jung T, Merker K et al (2004) Decreased proteolysis caused by protein aggregates inclusion bodies, plaques, lipofuscin, ceroid, and “aggresomes” during oxidative stress, aging, and disease. Int J Biochem Cell Biol 36:2519–2530
Riga D, RIGA S, Halalau F et al (2006) Brain Lipopigment accumulation in normal and pathological aging. Ann N Y Acad Sci 1067:158–163
Gonzáles-Scarano F, Baltuch G (1999) Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci 22:219–240
Keller JN, Dimayuga E, Chen Q et al (2004) Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain. Int J Biochem Cell Biol 36(12):2376–2391
Moreira PI, Carvalho C, Zhu X et al (2010) Mitochondrial dysfunction is a trigger of Alzheimer’s disease pathophysiology. Biochim Biophys Acta 1802(1):2–10
Good NE, Winget GD, Winter W et al (1966) Hydrogen ion buffers for biological research. Biochemistry 5(2):467–477
Montanaro J, Gruber D, Leisch N (2016) Improved ultrastructure of marine invertebrates using non-toxic buffers. PeerJ 4:e1860
Nagai T, Yokomori H, Yoshimura K (2004) Actin filaments around endothelial fenestrae in rat hepatic sinusoidal endothelial cells. Med Electron Microsc 37(4):252–255
Pegg DE (2007) Chapter 3: Principles of cryopreservation. In: Day JG, Stacey GN (eds) Cryopreservation and freeze-drying protocols, 2nd edn, pp 39–59. ISBN: 1-58829-377-7
Clancy B, Cauller LJ (1998) Reduction of background autofluorescence in brain sections following immersion in sodium borohydride. J Neurosci Methods 83(2):97–102
Craig AS (1974) Sodium borohydride as an aldehyde blocking reagent for electron microscope histochemistry. Histochemistry 42(2):141–144
Watanabe S, Punge A, Hollopeter G et al (2011) Protein localization in electron micrographs using fluorescence nanoscopy. Nat Methods 8(1):80–84
Mao S-Y (2012) Chapter 3: Conjugation of fluorochromes to antibodies. In: Javois LC (ed) Methods in molecular biology, vol 115: Immunocytochemical methods and protocols, 2nd edn. Humana Press Inc., Totowa, NJ
Guntern R, Vallet P, Bouras C et al (1989) An improved immunohistostaining procedure for peptides in human brain. Experientia 45:159–161
Brorson SH (1997) Bovine serum albumin (BSA) as a reagent against non-specific immunogold labeling on LR-White and epoxy resin. Micron 28(3):189–195
Acknowledgments
We would like to specially acknowledge Dr. Alberto Rábano, director of the CIEN Foundation tissue bank (BT-CIEN) for the tissue samples.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kun, A., González-Camacho, F., Hernández, S., Moreno-García, A., Calero, O., Calero, M. (2018). Characterization of Amyloid-β Plaques and Autofluorescent Lipofuscin Aggregates in Alzheimer’s Disease Brain: A Confocal Microscopy Approach. In: Sigurdsson, E., Calero, M., Gasset, M. (eds) Amyloid Proteins. Methods in Molecular Biology, vol 1779. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7816-8_31
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7816-8_31
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7815-1
Online ISBN: 978-1-4939-7816-8
eBook Packages: Springer Protocols