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Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease
Published on: April 19, 2021
Lipofuscin autofluorescence confounds intracellular amyloid β detection in the aged mouse brain
Godfried Dougnon1, Hideaki Matsui1
1Department of Neuroscience of Disease, Brain Research Institute, Niigata University, Niigata 951-8585, Japan.
Abstract:
Intracellular amyloid β (Aβ) accumulation is a contentious feature of Alzheimer's disease (AD), increasingly reported in young adults and aged animal models of AD. However, autofluorescent lipofuscin granules which consist of a mixture of highly oxidized lipids, misfolded proteins, and metals, accumulates with aging in neurons and microglia and renders difficult the interpretation of immunofluorescence-based studies. Here, we show that lipofuscin accumulation in aged wild-type (WT) mouse brains exhibits significant spectral overlap with commonly used antibodies for Aβ detection, leading to potential misinterpretation of intracellular Aβ signals. Through a combination of dye staining, immunohistochemistry (IHC), and confocal microscopy, we show that fluorescence signals resembling intracellular Aβ and commonly reported in aged animal models of AD, may reflect the presence of lipofuscin granules. Importantly, these signals persisted in control sections where primary Aβ antibodies were omitted, but disappeared following TrueBlack autofluorescence quencher. We also performed Aβ immunofluorescence staining using 5xFAD mice as model for AD, revealing that intracellular Aβ in these models can be diminished by TrueBlack treatment, thus confounding the interpretation of true intracellular Aβ signals. Our findings underscore the need for caution in interpreting intracellular Aβ signals in young adults and aged models of Aβ pathology inside neurons or microglia.
Insights
Lipofuscin autofluorescence can mimic intracellular amyloid-beta (Aβ) signals in Alzheimer
Area of Science:
- Neuroscience
- Alzheimer's Disease Research
- Cellular Biology
Background:
- Intracellular amyloid-beta (Aβ) accumulation is a debated hallmark of Alzheimer's disease (AD).
- Aging leads to lipofuscin accumulation in neurons and microglia, potentially interfering with Aβ detection.
- Autofluorescence from lipofuscin can complicate immunofluorescence studies for Aβ.
Purpose of the Study:
- To investigate whether lipofuscin autofluorescence can be mistaken for intracellular Aβ signals.
- To assess the impact of lipofuscin on Aβ detection in aged wild-type (WT) and 5xFAD mouse models.
- To evaluate the efficacy of autofluorescence quenchers in resolving Aβ signal misinterpretation.
Main Methods:
- Immunohistochemistry (IHC) and confocal microscopy were employed.
- Dye staining was used to identify lipofuscin granules.
- Autofluorescence quenching with TrueBlack was tested on aged WT and 5xFAD mouse brain sections.
Main Results:
- Lipofuscin granules in aged WT mouse brains showed spectral overlap with Aβ antibodies, mimicking intracellular Aβ.
- Aβ-like signals persisted in control sections lacking primary antibodies but were abolished by TrueBlack.
- TrueBlack treatment reduced apparent intracellular Aβ signals in 5xFAD mice, indicating lipofuscin interference.
Conclusions:
- Autofluorescent lipofuscin can lead to misinterpretation of intracellular Aβ signals in aged animal models.
- Caution is required when interpreting intracellular Aβ findings, especially in young adults and aged models.
- Autofluorescence quenching methods are crucial for accurate assessment of Aβ pathology in neurons and microglia.
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