Technical aspects of opioid receptor localization: detection of opioid receptor proteins by immunocytochemistry or with a biotinylated dynorphin analog

Abstract

Opioid receptors were localized at the cellular level, using either anti-opioid receptor antibodies or a biotinylated opioid ligand. In addition, a simple method was developed for selection of second antisera on their potencies to detect particular monoclonal antibodies (mAbs). Most anti-opioid receptor antibodies tested were not able to recognize the opioid receptor in frozen or fixed tissue sections, which was in contrast with their ability to recognize opioid receptors in isolated membrane fractions. However, one batch of anti-idiotypic mAbs gave a good immunocytochemical staining. Distribution of immunoreactivity suggested that these antibodies recognized more than one opioid receptor subtype. After very short fixation times, staining with a biotinylated kappa-selective ligand (DAKLIB) could be observed in the neural and intermediate lobe of pituitary. This binding could be displaced by non-biotinylated DAKLI. The pattern of DAKLIB staining in the neural lobe had the appearance of binding to both nerve fibres and astrocytes. The present results show successful staining of tissue sections with anti-idiotypic antibodies and with a biotinylated ligand. The specificity is discussed in the light of control experiments, pharmacological data and previous studies.