An immunohistochemical protocol for visualizing adrenergic receptor subtypes in the rhesus macaque hippocampus

Background: The noradrenergic system is an important modulatory system in the brain, and dysfunction in this system is implicated in multiple neurodegenerative diseases. The study of this system in neuronal tissues relies on the availability of specific antibodies but to date no protocol exists for immunohistological visualization of α1, α2, and β adrenergic receptors in rhesus macaques. New method: Here, we test the ability of various commercially available antibodies to detect these receptors in the primate brain and develop a protocol for visualization of receptors alongside noradrenergic axons and glial and vascular cells that interact with the noradrenergic system. Results: Of the eleven primary antibodies for adrenergic receptors tested, five did not produce staining at any concentration. The remaining six antibodies underwent a preadsorption protocol to determine specificity of the antibody to its’ immunogen sequence. Two antibodies failed this test, indicating they were binding to other targets in the brain. We then determined optimum concentrations for the remaining four antibodies. Additionally, we develop an immunofluorescence protocol that allows for the visualization of each AR - α1, α2a, or β1 – along with adrenergic axons as well as with glia and vasculature. Comparison with existing methods: While protocols exist for visualizing receptors in rodents, this is the first protocol for use in nonhuman primates. Conclusions: Seven out of the eleven tested antibodies were inaccurate, highlighting the importance of comprehensive testing. The stringent tests conducted here suggest that some commercially available antibodies can reliably detect adrenergic receptor subtypes in nonhuman primate tissue.