A fast and easy fluorescent counterstaining method for neuroanatomical studies by using Acridine Orange

Acridine Orange is commonly used as a fluorescent counterstain in fluorescent tract tracing techniques. Here we describe a method in which the substitution of the standard washing solutions (i.e., 0.9% saline) for a diluted solution of Acridine Orange (0.001%) during the perfusion of the animal before fixation provides a fluorescent counterstaining compatible with Fast Blue fluorescent retrograde labeling. In contrast to other fluorescent counterstaining methods, this procedure minimizes the diminution in the fluorescence of the tracer during the handling of sections.