Abstract
Actin is a key component of the cytoskeleton and also plays diverse roles within the cell nucleus. While polymerized F-actin can be detected using a wide range of probes, reliable methods to identify unpolymerized (“G-”) actin in fixed cells are relatively limited. Fluorescently labeled DNase I has long served as a high-affinity probe for monomeric actin and has recently gained renewed interest in nuclear actin research. Here, we briefly review established methods for visualizing actin in the cytoplasm and nucleus and revisit the history and molecular basis of the DNase I–actin interaction that forms the basis for DNase I staining. We then highlight practical considerations for interpreting DNase I fluorescence signals, such as its binding to filament pointed ends, the influence of divalent cations on its enzymatic activity and DNA binding, and protocol-dependent variations. Notably, fixation conditions significantly influence staining outcomes, affecting the distribution of nuclear versus cytoplasmic signals. Overall, this review aims to provide a concise framework for effectively using DNase I staining to monitor cellular pools of unpolymerized actin in a consistent and interpretable way.