Propidium iodide (or PI) is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used to stain DNA. It can be used to differentiate necrotic, apoptotic and normal cells. Propidium iodide (PI) binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per 4–5 base pairs of DNA. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining. Once the dye is bound to nucleic acids, its fluorescence is enhanced 20- to 30-fold, the fluorescence excitation maximum is shifted ~30–40 nm to the red and the fluorescence emission maximum is shifted ~15 nm to the blue. Although its molar absorptivity (extinction coefficient) is relatively low, PI exhibits a sufficiently large Stokes shift to allow simultaneous detection of nuclear DNA and fluorescein-labeled antibodies, provided the proper optical filters are used. PI is suitable for fluorescence microscopy, confocal laser scanning microscopy, flow cytometry, and fluorometry. PI is membrane impermeant and generally excluded from viable cells. PI is commonly used for identifying dead cells in a population and as a counterstain in multicolor fluorescent techniques. The counterstaining protocols below are compatible with a wide range of cytological labeling techniques—direct or indirect antibody-based detection methods, mRNA in situ hybridization, or staining with fluorescent reagents specific for cellular structures. These protocols can be modified for tissue staining. A typical use of Propidium Iodide in Plant Biology is to stain the cell wall. Especially useful for Arabidopsis thaliana seedlings root tissue observed by confocal microscopy, it allows to reveal the outlines of cells in the root tip. This red fluorescent background is useful to determine the sub-localization of a gene of interest expressed as a Green Fluorescent Protein fusion.
