Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease
Summary
In the neuropathology of Alzheimer’s disease, one of the most crucial characteristics is the deposition of amyloid-β. In this protocol, we describe the method of immunofluorescent staining in 5×FAD transgenic mouse to detect amyloid-β accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.
Abstract
Alzheimer’s disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-β (Aβ) in the brain. Aβ is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. In pathological circumstances, the increased β-cleavage of APP leads to overproduction of Aβ, which aggregates into Aβ plaques. Since Aβ plaques are a characteristic of AD pathology, detecting the amount of Aβ is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize Aβ deposition. The mouse model used in our experiments is 5×FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study Aβ pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect Aβ accumulation in 5×FAD mice. With this protocol, researchers can investigate Aβ pathology in an AD mouse model.
Introduction
Alzheimer's disease (AD) is a neurodegenerative disease that causes 60%-70% dementia around the world and costs much social resources1. It is well-known that accumulation of amyloid-β (Aβ) is a pathological hallmark in Alzheimer's disease. Amyloid precursor protein (APP) is an integral membrane protein that exists in many tissues. Aβ peptide, consisting of 36-42 amino acids2, is produced by the subsequent cleavage of β- and γ-secretase in APP3,4. Changes in APP cleavage and mutations in APP gene lead to overproduction of Aβ. Aβ molecules can aggregate to form oligomers or fibrils, which are believed to be neurotoxic5,6. In previous studies, the accumulation of Aβ was demonstrated to be correlated with neuronal death in AD7,8,9.
The 5×FAD (C57BL/6J) transgenic mice contain 3 mutations in APP, and 2 mutations in PSEN1. The accumulation of intracellular Aβ starts as early as 1.5 month of age. Extracellular accumulation of Aβ was found around 2 months, in the cortex and hippocampus. The accumulation increased rapidly with age10. The well-documented Aβ pathology makes it a good animal model for our protocol.
The goal of the described staining method is to visualize and quantify Aβ deposition in the brain of AD mice model. The procedure including transcardial paraformaldehyde perfusion, cryosectioning, immunofluorescent staining and quantification to detect Aβ accumulation in 5×FAD mice will be introduced. This protocol is a reliable and easy method to investigate Aβ pathology in AD mouse model.
Protocol
Representative Results
Discussion
Immunofluorescent staining using 6E10 antibody can specifically detect Aβ accumulation in the brain, which is easy to be quantified by Image J. What is worth noticing is that some crucial steps in this protocol may affect the results.
To prevent slices from peeling off or breakage shown in Figure 3C, a few key points should be noticed. Perfusion should proceed rapidly after making the incision on the diaphragm because of the irreversible pathophysiological ef…
Declarações
The authors have nothing to disclose.
Acknowledgements
This research was supported by a general project of Natural Science Foundation of China (grant number: 81974157) from the Natural Science Foundation of China, a Jiangsu Special Appointed Professorship (to C.L.) from Jiangsu Education Department, a Jiangsu Province Innovative and Entrepreneurial Team Program (to H.Z., A. L., W.W., C.L. and Y.S.), a starting grant of excellent talent (D2019025) and Innovation and Entrepreneurship Training Program (201910313038Z to Z.S., Y.X., M.Z. and J.D.) from Xuzhou Medical University. This research was also supported by National Demonstration Center for Experimental Basic Medical Science Education (Xuzhou Medical University).
Materials
| Anesthetia | |||
| Injection syringe | KLMEDICAL | 1 mL | |
| Pentobarbitual sodium | Sigma-Aldrich | P3761 | 1% in saline for i.p. injection |
| Thoracotomy: | |||
| IRIS-Fine Straight iris scissors (~11.5 cm) | RWD | S12010-11 | |
| Sharp curved surgical scissors(11.5 cm) | RWD | S14016-11 | |
| Straight dissecting forceps (~10.5 cm) | RWD | F12010-10 | |
| Perfusion | |||
| Centrifugal tube (5 mL) | Biosharp | ||
| Injection syringe | KLMEDICAL | 20 mL | |
| Paraformaldehyde | Vicmed | VIH100 | |
| PBS 0.01M (PH7.2-7.4) powder | Vicmed | VC2001P | Add deionized water to make solution |
| Fixation, Dehydration and Cryosectioning | |||
| Adhesion microscope slides | CITOTEST | 188105 | 76.2 mm × 25.4 mm |
| Microtome Cryostat | LEICA | CM1950 | |
| Optimal cutting temperature compound (OCT) | Sakura Finetek | 4583 | |
| Sucrose | VETEC | V900116 | |
| Immunofluorescence staining | |||
| Fluorescence microscope | OLYMPUS | IX-81 | |
| Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 | Thermo Fisher Scientific | A-11005 | 200X |
| Image-Pro Plus 7.0C | Media Cybernetics | Scientific graphing and data analysis software | |
| Immunohistochemical Wet Box (black) | Sinylab | Customized | 300 mm × 100 mm × 38 mm, groove depth 27 mm, can contain 10 standard slides |
| Pipet | Thermo Scientific | ||
| Pipette tips | Well-offer | ||
| Plastic staining box | Sinylab | Customized | 30 mL, can contain 5 standard slides |
| Primary Antibody Dilution Buffer | made in our lab | 1% BSA 1g, 0.3% Triton X-100 300ul, 0.01% sodium azide 10mg in 100ml PBS | |
| Purified anti beta amyloid,1-16 antibody (6E10) | Biolegend | SIG-39320 | 500X |
| Quick Antigen Retrieval Solution for Frozen Sections | KeyGEN BioTECH | KGIHC005 | 5X |
| Quantification | |||
| Graphpad Prism 8.0.1 | Graphpad | Medical mapping software | |
| Image J Fiji 2.0.0 | National Institute of Health | Scientific graphing and data analysis software | |
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