Multiple immunofluorescent labelling combined with confocal, or confocal and electron microscopy makes it possible to determine the precise cellular localization and coexistence of different molecules. Although the theories of these methods are very simple, the success of these techniques depends on several factors, such as fixation, antibody selection and embedding protocols. Endomorphin-2 is an endogenous ligand of the mu opioid receptor that has spinal analgesic effect in different nociceptive modalities. E2-like immunoreactivity is present mainly in primary afferents in the spinal dorsal horn, but its biosynthetic pathway(s) is (are) still unknown. We have pharmacological evidence for that membrane-bound dipeptidyl peptidase IV (DPP4) enzyme may be switched into "synthase" functional mode by the DPP4 inhibitor Ile-Pro-Ile in depolarization sensitive manner and generates E2 extracellularly.

In this study, we will demonstrate the methodological aspect of the work in which we have looked for morphological evidence for the extracellular generation of E2. We will also show the advantages and the drawbacks of the available quantitative analysis to produce not only qualitative but also quantitative data.