DOI: 10.5061/dryad.brv15dvkn
title: Immunohistochemistry method for measuring autophagy flux using MAP1LC3/LC3 and SQSTM1 as core markers

description: 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 enables reliable
 autophagy quantification using a standard light microscope, bridging the
 gap between experimental research and clinical diagnostics. Our protocol
 allows accurate autophagy evaluation in fixed tissues, offering practical
 applications in biomedical research and clinical pathology assessment.
description: <strong>Day 1</strong>
 <strong>1.     </strong><strong>De-paraffinize
 and rehydrate slides.</strong> Carry out the following exchange at
 room temperature, manually in Coplin jars (Millipore-Sigma, S5641), or
 using an automated embedding system. <strong>See Notes and Tips
 3.</strong> ·       Warm the slides at
 60<sup>o</sup>C 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). <strong>2.    
 </strong><strong>Heat-induced antigen
 retrieval</strong>: ·       Pre-warm citrate
 buffer in a polypropylene Coplin jar. <strong>See Notes and Tips
 4.</strong> ·       Place the slides in the
 Coplin jar. ·       Place the Coplin jar in a boiling
 water bath for 30 min. ·       Remove the Coplin jar
 from the boiling water. ·       Allow to cool slowly at
 room temperature for 20 min. <strong>3.    
 </strong><strong>Blocking of endogenous proteins.
 </strong>Blocking steps are crucial in IHC to prevent excessive
 background staining in images. <strong>See Notes and Tips
 5.</strong> ·       Remove slides from the
 citrate buffer one by one and dip quickly in double-distilled
 H<sub>2</sub>O. ·       Gently blot on a
 paper tissue to remove liquid. ·       Circle the
 tissue sections with an ImmEdge pen (Vector, H-4000). 
 ·       Immediately cover the tissue sections with PBS.
 <strong>Note</strong>: Do not allow the sections to
 dry. ·       Apply PBS to the sections for 5 min to
 wash (3X). ·       Apply the blocking solution to the
 sections for 30 min. ·       Apply PBS to the sections
 for 5 min to wash (3X). <strong>Note:</strong> If you are
 using an antibody produced in the same species as your tissue (e.g., a
 mouse antibody on mouse tissue), blocking endogenous IgG is necessary. For
 example, use goat anti-mouse IgG Fab fragment (Jackson ImmunoResearch,
 115-007-003) diluted 1:10 in PBS for a minimum of 1 h. It is best to
 incubate overnight at 4°C. Then, wash sections in PBS for 5 min (3X). If
 this is not an issue, proceed to the next step. ·      
 Apply freshly prepared 3% H<sub>2</sub>O<sub>2
 </sub>(in PBS) for 10 min. This step is necessary to eliminate
 endogenous hydrogen peroxidases. <strong>Note:</strong> The
 incubation time may have to be increased to 15 or 20 min if the tissue has
 a lot of blood. ·       Apply PBS to the sections for 5
 min to wash (3X). ·       Apply avidin blocking
 solution for 15 min. <strong>Note</strong>: This solution
 works just as well when diluted in an equal volume of PBS.
 ·       Apply PBS to the sections for 5 min to wash
 (3X). ·       Apply biotin-blocking solution for 15
 min. <strong>Note</strong>: This solution works just as well
 when diluted in an equal volume of PBS. ·       Apply
 PBS to the sections for 5 min to wash (3X).
 <strong>4.    
 </strong><strong>Immunostaining:</strong>
 ·       Arrange sections in a moist chamber to avoid drying of
 tissues. ·       Apply primary antibody and incubate
 overnight at 4°C. <strong>Note:</strong> Leave one section
 without primary antibody, and cover only with blocking solution to be used
 as a negative control. <strong>See Notes and Tips
 7.</strong>
 <strong><em> </em></strong><strong>Day 2</strong> ·       Apply PBS to the sections for 5 min to wash (3X). ·       Apply biotinylated secondary antibody (from the appropriate organism corresponding to the primary antibody) to all sections (including the negative control) for 30 min. ·       Apply PBS to the sections for 5 min to wash (3X). ·       Apply activated “ABC” solution for 30 min. ·       Apply PBS to the sections for 5 min to wash (3X). ·       Apply freshly prepared DAB substrate to the sections for up to 2 min. <strong>See Notes and Tips 8.</strong> ·       Stop reaction by immersing slide in double-distilled H<sub>2</sub>O in a Coplin jar. ·       Wash slides with H<sub>2</sub>O in a Coplin jar (3X). ·       Counterstain with Mayer hematoxylin for 1-4 min. <strong>See Notes and Tips 9.</strong> ·       Wash slides with H<sub>2</sub>O in a Coplin jar (3X). ·       Immerse in 2% sodium bicarbonate solution, pH 8 for 20 s. ·       Wash slides with H<sub>2</sub>O in a Coplin jar (3X). <strong>5.     </strong><strong>Dehydration and mounting. </strong>Dehydration is done by reversing the steps of hydration. Carry out the following exchange at room temperature, manually in Coplin jars, or using an automated embedding system. <strong>See Notes and Tips 10.</strong> ·       Place the slides in 70% ethanol for 1 min. ·       Place the slides in 95% ethanol for 1 min. ·       Place the slides in 100% ethanol for 1 min (2X). ·       Place the slides in xylene for 5 min (3X). ·       Remove slides one by one. ·       Blot the edge of the slide to a paper tissue to remove the extra liquid. ·       Add one drop of mounting medium on the top of the tissue section. ·       Mount a coverslip on the mounting medium. ·       Gently press the coverslip with the tip of a pencil to remove  any possible bubbles. ·       Lightly clean the extra mounting agent surrounding the coverslip using the edges of a paper tissue. ·       Leave slides at room temperature overnight to ensure they are completely dry. <strong>Day 3 </strong> <strong>Imaging </strong> To perform microscopy imaging for DAB IHC, tissue sections are prepared and stained with primary and secondary antibodies, followed by a DAB substrate, then mount the stained sections on slides and cover them with coverslips as described above. Use a bright-field microscope equipped with a camera. Adjust the light and focus to visualize the brown DAB precipitate. Capture images at multiple magnifications (e.g., 10x, 20x, 40x). Ensure consistent imaging settings for all samples. Save and label images appropriately for analysis. This protocol provides clear visualization of DAB-stained tissue sections, facilitating accurate evaluation and documentation. <strong>Day 4</strong> <strong>Scoring </strong> To ensure unbiased and accurate results, scoring DAB IHC for LC3B and SQSTM1 puncta involves a meticulous evaluation by three pathologists in a blinded manner. Each pathologist independently examines stained tissue sections under a microscope, focusing on the presence and intensity of the brown DAB residue, which indicates the presence of the target antigens LC3B and SQSTM1. The scoring process includes assessing the number of puncta staining and the percentage of positively stained cells. The puncta numbers for LC3B and SQSTM1 is graded on a scale from 0 to 3, where 0 indicates no puncta staining (null), 1 indicates low number of puncta staining (low), 2 indicates moderate numbers of puncta staining (medium), and 3 indicates high numbers of  puncta staining (high). The percentage of positively stained cells is categorized into ranges (e.g., 0-25%, 26-50%, 51-75%, and 76-100%). Each pathologist assigns scores independently, unaware of the others' assessments or identifying information about the samples. This blinded approach minimizes bias and enhances the reliability of the results. After scoring, the results from the three pathologists are compared and averaged to obtain a final score for each sample. This rigorous evaluation method ensures that the interpretation of LC3B and SQSTM1 puncta via DAB-based IHC staining is accurate and reproducible, providing reliable data for research and diagnostic purposes. This process is critical for studies investigating autophagy, as LC3B and SQSTM1 are key markers of this cellular process. <strong>Day 5 </strong> <strong>Autophagy Flux Evaluation, Overview</strong> Evaluating autophagy flux involves assessing the expression levels and patterns of cytosolic LC3B and SQSTM1 puncta, which are crucial markers of autophagy activity. Autophagy flux refers to the dynamic autophagosome formation, maturation, and degradation process. Note that there is an ongoing balance between LC3B and SQSTM1 synthesis and degradation. During autophagy induction, LC3B levels in particular increase, although the extent can be tissue- and cell-type specific. LC3B is present on both sides of the phagophore, the initial sequestering compartment, and the autophagosome. The LC3B on the outer surface of the autophagosome is removed and recycled, whereas the population on the inner surface is exposed to the lysosome lumen and degraded, lowering the LC3B level. SQSTM1 is bound to LC3B on the inner surface of the phagophore and autophagosome and hence is also degraded in the lysosome. Therefore, an increase in LC3B can reflect A) autophagy induction or B) a block in autophagosome-lysosome fusion or degradation within the lysosome. Accordingly, measuring LCB3 alone is not sufficient to assess autophagic flux. In general, an accumulation of LC3B along with SQSTM1 would indicate a block in flux. In contrast, an increase in LC3B accompanied by a decrease in SQSTM1 could indicate high autophagy activity (a large increase in LC3B due to autophagy induction along with turnover of both proteins). Thus, the balance between the levels of LC3B and SQSTM1 provides substantial information about flux.
description: # 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**: Continuous number of sample * **Cytosolic Dot P62**: IHC staining score for **P62 (SQSTM1) puncta**, a marker of **autophagic cargo accumulation**. High P62 levels suggest **autophagy inhibition**, while low levels indicate **efficient cargo degradation**. **2. File Name:** **LC3.csv** This dataset presents **immunohistochemistry (IHC)-based autophagy flux assessment**, focusing on key autophagy markers: **LC3**. 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 LC3 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**: Continuous number of sample * **Cytosolic Dot LC3**: IHC staining score for **LC3 puncta**, reflecting the presence of **autophagosomes**. High LC3 expression suggests **autophagosome accumulation**, which may indicate either **active formation or impaired clearance**. **3. File Name:** **Autophagy Flux.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 and LC3 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**: Continuous number of sample * **Cytosolic Dot P62**: IHC staining score for **P62 (SQSTM1) puncta**, a marker of **autophagic cargo accumulation**. High P62 levels suggest **autophagy inhibition**, while low levels indicate **efficient cargo degradation**. * **Cytosolic Dot LC3**: IHC staining score for **LC3 puncta**, reflecting the presence of **autophagosomes**. High LC3 expression suggests **autophagosome accumulation**, which may indicate either **active formation or impaired clearance**. * **Autophagy Flux**: A **functional interpretation** of autophagy status based on the combined expression of **LC3 and P62**, allowing for an assessment of **autophagy activity versus inhibition**. **4. Interpreting Autophagy Flux Using IHC** IHC-based autophagy flux evaluation relies on the **co-expression patterns of P62 and LC3**: * **High LC3 & High P62** **Autophagy flux inhibition** (autophagosome accumulation without degradation). * **High LC3 & Low P62** **Very active autophagy** (efficient cargo degradation). * **High LC3 & Null P62** **Excessive autophagosome formation** with continuous turnover. * **Medium LC3 & Null P62** **Very active autophagy flux**. * **Low LC3 & Null P62** **Highly active autophagy** with minimal detectable substrate accumulation. * **Low LC3 & Low P62** **Strong autophagic clearance of cellular components**. * **Null LC3 & Low P62** **Maximal autophagic degradation**. **Scoring System for IHC Staining Intensity:** * **High (3)**: Strongly positive staining and high numbers of puncta. * **Medium (2)**: Moderately positive staining and medium numbers of puncta. * **Low (1)**: Weakly positive staining and low number of puncta . * **Null (0)**: No detectable staining and puncta structure. **5. Applications and Research Impact** This dataset serves as a **practical tool for evaluating autophagy flux in formalin-fixed tissues**, making it applicable to **cancer, fibrosis, neurodegeneration, and metabolic diseases**. By integrating **LC3 and P62 expression**, researchers can distinguish between **autophagy activation, inhibition, and defective flux**, helping to refine **therapeutic strategies targeting autophagy modulation**. **6. Conclusion and Future Directions** This **IHC-based approach to autophagy flux assessment** bridges the gap between **molecular pathology and mechanistic autophagy research**. The dataset is valuable for **histopathological studies**, **biomarker discovery**, and **therapeutic target validation**. **7. File Name:** **LC3** This file contains **high-resolution tissue microarray (TMA) images** **phase-contrast microscopy (40 X)**, specifically immunostained for **LC3 (Microtubule-Associated Protein 1 Light Chain 3)**. LC3 is a **key marker of autophagy**, playing a crucial role in **autophagosome formation and turnover**. The **localization of LC3 puncta** within the cytoplasm provides insights into **autophagic activity and flux** in various cancer and pathological tissue samples. Each **folder** identifies sample localization using the row name and column number in tissue microarray slide (e.g., **"A1" refers to row A, column 1**). * **Staining Overview:**\ The TMA slides are **immunostained for LC3**. The **distribution of LC3 puncta** helps in assessing whether autophagy is **activated or impaired** in different pathological conditions. * **Phase-Contrast Imaging:**\ The **phase-contrast microscopy** technique enhances **cellular structures without requiring additional dyes**, providing clear visualization of **LC3 localization within autophagic vesicles** and **cytoplasmic compartments**. - **Contents:**\ The file contains TMAs. Each sample is labeled with its **name of protein** and corresponding **localization in microarray slide**. **8. File Name:** **p62-SQSTM1** This file contains **high-resolution tissue microarray (TMA) (40X)**, specifically immunostained for **p62/SQSTM1 (Sequestosome 1)**. **p62 is a crucial autophagy adaptor protein**, responsible for **selecting and delivering ubiquitinated proteins to autophagosomes**. The **accumulation or degradation of p62** is a widely used indicator of **autophagic flux and efficiency**. Each **folder** identifies sample localization using the row name and column number in tissue microarray slide (e.g., **"A1" refers to row A, column 1**). * **Staining Overview:**\ The TMA slides are **immunostained for p62**, highlighting its expression and accumulation. **Cytoplasmic dot-like staining** represents **p62 accumulation**, which often correlates with **autophagy inhibition**. * **Phase-Contrast Imaging:**\ The **phase-contrast imaging technique** enhances **cellular structures and protein localization** without additional staining, providing clear visualization of **p62 puncta accumulation or clearance** across different tissue conditions. * **Contents:**\ The file contains TMAs. Each sample is labeled with its **name of protein** and corresponding **localization in microarray slide**.