Counterstaining improves visualization of the myenteric plexus in immunolabelled whole-mount preparations.

Immunocytochemistry has emerged as a powerful research tool in neurobiology. One of the widely used methods is an indirect fluorescence technique that uses FITC- conjugated IgG to visualise protein expression within tissues, but a major drawback of this technique is the high background fluorescence due to non-specific antibody binding. Gut innervation is complex and best visualized in three-dimensions in whole mount preparations. We describe a simple and easy to use counterstaining procedure in conjunction with an indirect immunofluorescence technique in gut whole mount preparations that largely eliminates background fluorescence and creates a contrasting background against the bright antigen-antibody complexes. Furthermore, this technique allows the detailed qualitative and quantitative study of myenteric plexuses in whole-mount preparations.