Autophagy Analysis: A Step-by-Step Simple Practical Guide for Immunofluorescence and Western Blotting

Autophagy is a vital cellular process responsible for breaking down faulty cellular components and organelles, ultimately routed through lysosomes for degradation. This intricate mechanism involves the translocation of LC3, a cytoplasmic protein, onto the autophagosome membranes. As a result, it becomes feasible to discern cells engaged in autophagy by employing fluorescent markers designed for LC3 or other indicative autophagy markers. Although a variety of techniques such as immunofluorescence and western blotting serve as indispensable tools for assessing autophagy, the definitive confirmation comes from the visualization of autophagosomes using transmission electron microscopy. While numerous protocols for antibody staining can be found in scientific literature and on antibody suppliers’ websites, these procedures often demand significant time and financial resources for setup. This chapter endeavors to provide a user-friendly and cost-effective guide for practitioners seeking proficiency in immunofluorescence staining and western blotting techniques.