Fluorescent staining of nucleolar organizer regions for three-dimensional display by confocal laser scanning...

A new method of fluorescent staining of nucleolar organizer regions (F1NORs) is described. Aluminum ammonium sulfate was used instead of silver as the cationic metal ion for binding with NORs-associated proteins, and fluorescent morin was successively bound to aluminum by chelating with modification of the method developed by Malinin (1978). After bleaching the fluorescent staining of NORs by washing water, ordinary AgNORs staining was performed on the same section, and both images of F1NORs and AgNORs were found to coincide with each other. F1NORs staining of human malignant and benign tumors, and colorectal adenomas of borderline malignancy were examined by three-dimensional analysis of the fluorescence images under confocal laser scanning microscope (CLSM). A remarkable increase of F1NORs was found, not only in number but also in volume, with bizarre configuration in the process of tumor progression, and the F1NORs-CLSM technique may be helpful for daily pathological diagnosis of malignancy.