Immunohistochemistry method for measuring autophagy flux using MAP1LC3/LC3 and SQSTM1 as core markers

Macroautophagy/autophagy is a crucial cellular process for degrading and recycling damaged proteins and organelles, playing a significant role in diseases such as cancer and neurodegeneration. Evaluating autophagy flux, which tracks autophagosome formation, maturation, and degradation, is essential for understanding disease mechanisms. Current fluorescence-based methods are resource-intensive, requiring advanced equipment and expertise, limiting their use in clinical laboratories. Here, we introduce a non-fluorescent immunohistochemistry (IHC) method using MAP1LC3/LC3 and SQSTM1 as core markers for autophagy flux assessment. LC3 levels reflect autophagosome formation, whereas SQSTM1 degradation and a decrease in the number of its puncta indicate active flux (i.e., lysosomal turnover). We optimized chromogenic detection using diaminobenzidine (DAB) staining and developed a scoring system based on puncta number and the percentage of stained cells. This accessible, cost-effective method en..., Day 1
1.     De-paraffinize and rehydrate slides. Carry out the following exchange at room temperature, manually in Coplin jars (Millipore-Sigma, S5641), or using an automated embedding system. See Notes and Tips 3.
·       Warm the slides at 60oC for 30 min in the Coplin jar.
·       Place the slides in xylene for 5 min (3X), being sure to cover the samples in this and all subsequent steps.
·       Place the slides in 100% ethanol for 1 min (2X).
·       Place the slides in 95% ethanol for 1 min.
·       Place the slides in 70% ethanol for 1 min.
·       Wash with running tap water for 1 min.
·       Place the slides in PBS for 1 min (3X).
2.     Heat-induced antigen retrieval:
·       Pre-warm citrate buffer i..., , # Tissue Microarray

[https://doi.org/10.5061/dryad.brv15dvkn](https://doi.org/10.5061/dryad.brv15dvkn)

### **Description of the data and file structure**

*It is Tissue microarray data*

**Code/software**

Any software for image (adobe photoshop, image j) and excel/csv.

**1. File Name:**

**P62(SQSTM1).csv**

This dataset presents **immunohistochemistry (IHC)-based autophagy flux assessment**, focusing on key autophagy markers: **P62 (SQSTM1)**. Unlike fluorescence-based techniques, IHC offers a **non-fluorescent approach** to evaluating **autophagic activity in formalin-fixed, paraffin-embedded (FFPE) tissues**, making it a powerful tool for retrospective pathological analyses. The dataset enables **quantitative scoring of P62 puncta**, providing insights into **autophagy flux status** in various tissue contexts.

**Structure of the Dataset**

The dataset consists of the following key variables:

* **Pos.**: Position identifier in the **tissue microarray (TMA)**.
* **No**: Continuou...,

巨自噬（Macroautophagy，又称自噬（autophagy））是降解和回收受损蛋白质与细胞器的关键细胞过程，在癌症、神经退行性疾病等多种疾病中发挥重要作用。评估自噬流——即追踪自噬体的形成、成熟与降解过程——对于阐明疾病发病机制至关重要。当前基于荧光的检测方法资源消耗极大，需要高端设备与专业技术知识，限制了其在临床实验室中的应用。本文介绍了一种以MAP1LC3/LC3和SQSTM1为核心标记物的非荧光免疫组织化学（immunohistochemistry，IHC）方法，用于自噬流评估。LC3水平可反映自噬体的形成，而SQSTM1的降解及其点状聚集数量减少则提示自噬流活跃（即溶酶体周转过程正常）。我们优化了基于二氨基联苯胺（diaminobenzidine，DAB）染色的显色检测方案，并开发了基于点状聚集数量与染色细胞百分比的评分系统。该便捷且具成本效益的方法……（原文此处截断） Day 1
1. 玻片脱蜡与复水：室温下于Coplin染色缸（Millipore-Sigma，货号S5641）中手动完成以下试剂更换流程，或使用自动化包埋系统操作。详见注意事项与技巧3。
· 将玻片置于Coplin缸中，60℃预热30分钟。
· 将玻片置于二甲苯中孵育5分钟，重复3次；本步骤及后续所有步骤均需确保样本完全浸没于试剂中。
· 将玻片置于100%乙醇中孵育1分钟，重复2次。
· 将玻片置于95%乙醇中孵育1分钟。
· 将玻片置于70%乙醇中孵育1分钟。
· 用流动自来水冲洗玻片1分钟。
· 将玻片置于磷酸盐缓冲液（PBS）中孵育1分钟，重复3次。
2. 热诱导抗原修复：
· 预温柠檬酸盐缓冲液……（原文此处截断）

# 组织微阵列
[https://doi.org/10.5061/dryad.brv15dvkn](https://doi.org/10.5061/dryad.brv15dvkn)

### 数据与文件结构说明
本数据集为组织微阵列数据。

**代码与软件**
可使用任意图像处理软件（如Adobe Photoshop、Image J）及Excel/CSV工具。

**1. 文件名：**
**P62(SQSTM1).csv**
本数据集聚焦于基于免疫组织化学（IHC）的自噬流评估，核心检测指标为关键自噬标记物P62（SQSTM1）。与基于荧光的检测技术不同，免疫组织化学法可对福尔马林固定石蜡包埋（formalin-fixed, paraffin-embedded，FFPE）组织的自噬活性进行非荧光检测，是回顾性病理分析的有力工具。本数据集可实现P62点状聚集的定量评分，为多种组织背景下的自噬流状态提供分析依据。

**数据集结构**
本数据集包含以下核心变量：
* **Pos.：** 组织微阵列（Tissue Microarray, TMA）中的位置标识符。
* **No：** 连续编号……