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 APP^3,4. Changes in APP cleavage and mutations in APP gene lead to overproduction of

Protocol

All experimental procedures were performed with the approval of the Institutional Animal Care and Use Committee of Xuzhou Medical University and in accordance with the guidelines of the Chinese governmental regulations for the care and use of laboratory animals.

1. Perfusion of Mice

NOTE: More details of the perfusion procedure can refer to the video from Wiliam Shain's lab ¹¹.

1. Anesthetize 5×FAD (C57BL/6J) transgenic mice with 1% pentobarbital sodium (50 mg/kg body weight) by intraperitoneal injection^12,13. Los

Results

We used the above-described immunofluorescent staining procedures to investigate the deposition of Aβ accumulation in 5×FAD mice of different age. Figure 3 represents typical results and suboptimal results using our protocol. Brain slices of 5-month and 8-month heterozygous 5×FAD transgenic mice and 4 to 6-month wild-type control were stained with 6E10 antibody and detected under a fluorescence microscope. Figure 3A shows Aβ accumulation in p...

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...

Materials

Name	Company	Catalog Number	Comments
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