Microwave staining of intestinal whole-mount preparations with Cuprolinic blue

Encouraged by the knowledge that microwaves have a beneficial effect on immunohistochemical reactions, the present study aimed to find out whether microwaves could improve the Cuprolinic Blue staining of enteric neurons as well as the actual method that has been developed for gastrointestinal whole-mount preparations. In addition to incorporating a microwave application in the method described by Holst and Powley (1995), some other modifications were made: two incubations before incubation in the staining solution and free-floating incubations. In the whole-mount preparations, most, if not all, enteric neurons were stained by Cuprolinic Blue. These neurons appeared as blue-green cells with non-reacting nuclei and neuronal processes. At higher magnification, the cytoplasm was characterized by a fine blue-green granulation, and the nucleolus in the nucleus appeared as a blue iridescent structure. Non-specific staining occurred in fibrocytes and epithelial cells but, because of their location and appearance, they could easily be distinguished from neurons. The modified incubations and the incorporation of a microwave application into the conventional Cuprolinic Blue staining method turn the method into an easy-to-use one that seems to visualize most, if not all, enteric neurons in whole-mount preparations of the pig jejunum.