In the presence of alkaline phosphatase, Permanent Red substrate produces a bright red precipitate at the site of reaction with the enzyme on tissues and cells. The precipitate produced is insoluble in alcohol and other organic solvents, so it is possible to dehydrate the slides with successive alcohol baths and to use an organic (non-aqueous) mounting medium.

This substrate is mainly used for immunohistochemistry and in situ hybridization techniques.

The appearance of the staining can be followed by observation with an optical microscope but also with a fluorescence microscope since the precipitate produced emits fluorescence in the infrared (it is necessary to use the same filters as for Rhodamine or Texas Red).