Use of acridine orange for histologic analysis of the central nervous system

The application of the fluorescent dye acridine orange (AO) to the staining of histologic sections of the brain, and its use for automatic cyto- and histophotometric evaluation are described and compared with the results obtained using cresyl violet. The most suitable procedure for aldehyde-fixed brain tissue, embedded in paraffin and sectioned at 5 micron, proved to be treatment of the sections with an aqueous solution of AO (1:50,000) at pH 1.2 for 30 min, followed by rinsing in distilled water for 10 min. This procedure revealed the morphology in a highly acceptable manner, clearly differentiating various cell components; its characteristics included exact reproducibility and high contrast. The degree of fading was calculable, with a very gradual decrease in fluorescent intensity. The AO procedure appears to be compatible with most other staining procedures that do not rely on the same binding mechanisms. Thus, AO staining has two advantages over the classical cytoarchitectural staining methods: first, it is more suitable for automated morphometric analysis, and second, it can be used in conjunction with immunologic and other techniques.