Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.