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.

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 60^oC 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 in a polypropylene Coplin jar. See Notes and Tips 4.

·       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.

3.     Blocking of endogenous proteins. Blocking steps are crucial in IHC to prevent excessive background staining in images. See Notes and Tips 5.

·       Remove slides from the citrate buffer one by one and dip quickly in double-distilled H₂O.

·       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. Note: 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). Note: 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% H₂O₂ (in PBS) for 10 min. This step is necessary to eliminate endogenous hydrogen peroxidases. Note: 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. Note: 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. Note: 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).

4.     Immunostaining:

·       Arrange sections in a moist chamber to avoid drying of tissues.

·       Apply primary antibody and incubate overnight at 4°C. Note: Leave one section without primary antibody, and cover only with blocking solution to be used as a negative control. See Notes and Tips 7.

 Day 2

·       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. See Notes and Tips 8.

·       Stop reaction by immersing slide in double-distilled H₂O in a Coplin jar.

·       Wash slides with H₂O in a Coplin jar (3X).

·       Counterstain with Mayer hematoxylin for 1-4 min. See Notes and Tips 9.

·       Wash slides with H₂O in a Coplin jar (3X).

·       Immerse in 2% sodium bicarbonate solution, pH 8 for 20 s.

·       Wash slides with H₂O in a Coplin jar (3X).

5.     Dehydration and mounting. 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. See Notes and Tips 10.

·       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.

Day 3

Imaging

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.

Day 4

Scoring

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.

Day 5

Autophagy Flux Evaluation, Overview

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.