A non-fluorescent immunohistochemistry method for measuring autophagy flux using MAP1LC3/LC3 and SQSTM1 as core markers

Abstract

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.

Keywords: autophagometer; autophagy flux measurement; cellular homeostasis analysis; chromogenic detection; cost‐effective autophagy assay; non‐fluorescent immunohistochemistry.

Fig. 1

IHC analysis of autophagy flux in human brain tissue. Representative immunohistochemical staining of LC3B (A) and SQSTM1 (B) in tissue sections, indicating autophagy flux. LC3B staining shows high numbers of cytosolic puncta (score 3) (black arrow) with 75–100% positively stained cells, indicating high autophagosome formation. In contrast, SQSTM1 staining shows no cytosolic puncta staining (score 0) with 0–25% positively stained cells, reflecting the increase in LC3 puncta is due to active formation of autophagosomes. Based on these findings, the autophagy flux is categorized as “Very active autophagosome formation (II).” We used a 40× objective for taking the image.