Assessing Iron Deposition in the Brains of 5xFAD Mice by Perls'/DAB Staining

Shuning Sang; Chenhao Tian; Jiahui Ding; Suochen Pang; Chao Liu

doi:10.3791/67501

JoVE Journal
Neuroscience
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JoVE Journal Neuroscience

Assessing Iron Deposition in the Brains of 5xFAD Mice by Perls’/DAB Staining

通过 Perls'/DAB 染色评估 5xFAD 小鼠大脑中的铁沉积

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07:32 min

May 23, 2025

DOI: 10.3791/67501-v

Shuning Sang*¹, Chenhao Tian*¹, Jiahui Ding^1,2, Suochen Pang^1,3, Chao Liu¹

¹Jiangsu Province Key Laboratory of Anesthesiology, Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, NMPA Key Laboratory for Research and Evaluation of Narcotic and Psychotropic Drugs, School of Anesthesiology,Xuzhou Medical University, ²Department of Anesthesiology,the Eye & ENT Hospital of Fudan University Shanghai, ³Department of Anesthesiology, Shanghai Tenth People's Hospital,Tongji University School of Medicine

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Overview

This protocol outlines techniques for assessing the distribution and quantity of iron deposition in the brains of an Alzheimer’s disease (AD) mouse model, specifically using 8-month-old 5xFAD transgenic mice. It examines iron accumulation using Perls/DAB staining, comparing results with wild-type mice.

Key Study Components

Area of Science

Neuroscience
Histochemical analysis
Alzheimer's disease research

Background

Iron deposition is linked to neurodegenerative diseases.
The study compares AD mouse models with wild-type counterparts.
Previous studies suggest iron accumulation correlates with pathology in AD.
Perls/DAB staining provides a sensitive method for detecting iron.

Purpose of Study

To assess iron deposition in brain tissues of 5xFAD mice.
To evaluate differences in iron accumulation compared to wild-type mice.
To establish a reliable protocol for studying iron levels in AD models.

Methods Used

Utilized brain sections from 5xFAD and wild-type mice.
Key procedures included chemical reagent preparation, brain sectioning, and staining with Perls/DAB.
Involved fixation, immersion in sucrose, and cryosectioning of brain tissues.
Image capture and data analysis were performed to quantify staining results.

Main Results

High Perls/DAB staining signals were observed in the hippocampus and cortex of 5xFAD mice.
Weak signals were found in both 2-month and 8-month wild-type mice.
The study validates Perls/DAB staining for sensitive and specific iron detection in brain tissues.
Findings highlight the association of iron with A-beta plaques in AD pathology.

Conclusions

The study demonstrates effective methodologies for measuring iron accumulation in AD mouse models.
Perls/DAB staining enhances sensitivity and specificity for detecting iron in neurological contexts.
Implications include understanding the role of iron in Alzheimer's disease and related pathologies.

Frequently Asked Questions

What are the advantages of using the 5xFAD mouse model?

5xFAD mice are known to exhibit early and aggressive amyloid plaque formation, making them suitable for studying iron deposition related to Alzheimer's pathology.

How is the Perls/DAB staining procedure implemented?

The protocol involves preparing chemical reagents, sectioning the brain, and applying staining solutions to visualize iron deposits in brain tissues.

What types of data are obtained from this protocol?

The protocol provides quantitative data on the distribution of iron deposits in brain tissues, which can be analyzed for statistical significance between different mouse genotypes.

Can this method be adapted for other models or conditions?

Yes, while tailored for the 5xFAD model, the staining and analysis techniques can be adapted to other neurodegenerative models as needed.

What are some limitations of the Perls/DAB staining method?

Possible limitations include potential background staining and the need for careful reagent handling to avoid excessive staining or tissue damage.

该协议提出了评估组织（尤其是大脑）中铁沉积的分布和数量的方法。该方案详细介绍了样品制备、Perls/DAB 染色、图像捕获和数据分析的程序。

该协议的目标是描述如何评估 AD 小鼠模型大脑中铁沉积的数量和分布。在该方案中，我们使用 8 个月大的 5xFAD 转基因小鼠作为 AD 小鼠模型，并将它们与同龄的野生型小鼠进行比较。我们包括制备化学试剂、切开大脑、执行 Perls/DAB 染色和分析所得图像的程序的详细信息。

适当麻醉鼠标并检查麻醉和镇痛的深度。露出它的心脏，然后切开右心房。依次从左心室注射 20 毫升 PBS 和 4% PFA。

请注意，应观察固定震颤。斩首老鼠并提取它的大脑。然后，将大脑固定在 4% PFA 中 12 至 24 小时。

将大脑浸入 15% 和 30% 蔗糖中 12 至 24 小时。从中间矢状切开大脑，用任一半切开。将大脑安装在旋钮上，在大脑周围放一个锡箔环，并在其中加入 OCT 以嵌入大脑。

设置低温恒温器的截面厚度和温度。然后，切开大脑。如果部分折叠，请使用软画笔展开部分。

用 PBS 将每个切片收集到载玻片中，然后消除切片上的气泡。选择具有完整脑组织的载玻片，并将其放入装满 PBS 的塑料染色盒中。将离心箱放在旋转摇床上，以慢速放置 5 分钟，以彻底冲洗掉残留的 OCT 化合物。

制备 20 毫升 2% 亚铁氰化钾和等体积的 2% 盐酸。然后，将它们混合在 50 毫升离心管中。使用镊子将玻片固定在试管中，并将混合物在 60 摄氏度的预热水浴中孵育 30 分钟。

用 PBS 清洗载玻片，并用薄纸擦去多余的液体。将玻片平放在实验室工作台上，甚至使用移液管将 DAB 溶液涂抹在组织上。孵育 10 分钟以增强 Perls 染色。

去除过量的 DAB 溶液，然后用 PBS 洗涤 3 次冲洗组织。在分级酒精溶液和二甲苯中依次脱水切片，每次脱水 3 分钟。用中性口香糖和盖玻片覆盖切片。

然后，让它们在通风橱中干燥。打开显微镜。调整光源的亮度。

在 4 倍放大物镜下专注于大脑切片。移动显微镜载物台以聚焦于具有高 Perls/DAB 染色信号的区域，尤其是海马体和大脑皮层。并捕获图像。

以 10 倍放大率打开物镜图像，然后将图像格式转换为 8 位灰度。将图像的灰度值转换为 OD 值，然后用阈值函数覆盖 Perls/DAB 染色阳性区域。选择以下设置选项，最重要的是，选择 limit to threshold 以排除背景噪音。

最后，选择测量值以获得用于统计分析的结果。为了研究铁在 AD 小鼠模型中的分布和积累，我们对矢状脑切片进行了 Perls/DAB 染色。在 5xFAD 小鼠的海马体和皮层中观察到高 Perls/DAB 信号，尤其是在海马体的下部，而 2 个月和 8 个月野生型小鼠的大脑均表现出较弱的信号。

在 40 倍以上的放大倍率下，5xFAD 小鼠中的信号出现在 A-β 斑块样结构中，与以前的研究一致。这些结果证明了 Perls/DAB 作为铁检测的组织化学技术的有效性。我们还展示了两个染色失败的实例。

在这些情况下，过度覆盖或不适当的脱水会导致这种情况。Perls/DAB 染色比传统 Perls 染色提供更强的信号和更好的背景对比度，使铁检测更灵敏、更准确。此外，它还检测松散结合的蛋白质复合物中的三价铁。

铁（如血红蛋白）不会发生反应。这大大减少了红细胞中铁和血红蛋白引起的不需要的信号。总体而言，Perls/DAB 染色适用于需要内侧特异性和敏感性的动物实验和病理学检查。

它为研究人员提供了一种以更少的时间和成本来可视化和量化铁积累的方法。

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