title: Antibody testing for brain immunohistochemistry: brain immunolabeling for the cannabinoid CB₂ receptor.
abstract: The question of whether cannabinoid CB₂ receptors are expressed on neurons in the brain and under what circumstances they are expressed is controversial in cannabinoid neuropharmacology. While some studies have reported that CB₂ receptors are not detectable on neurons under normal circumstances, other studies have reported abundant neuronal expression. One reason for these apparent discrepancies is the reliance on incompletely validated CB₂ receptor antibodies and immunohistochemical procedures. In this study, we demonstrate some of the methodological problems encountered using three different commercial CB₂ receptor antibodies. We show that (1) the commonly used antibodies that were confirmed by many of the tests used for antibody validation still failed when examined using the knockout control test; (2) the coherence between the labeling patterns provided by two antibodies for the same protein at different epitopes may be misleading and must be validated using both low- and high-magnification microscopy; and (3) although CB₂ receptor antibodies may label neurons in the brain, the protein that the antibodies are labeling is not necessarily CB₂. These results showed that great caution needs to be exercised when interpreting the results of brain immunohistochemistry using CB₂ receptor antibodies and that, in general, none of the tests for antibody validity that have been proposed, apart from the knockout control test, are reliable.
authors.affiliation: Department of Pharmacology and Toxicology, School of Medical Sciences, University of Otago Medical School, P.O. Box 913, Dunedin, New Zealand.
authors.forename: Jean-Ha
authors.initials: JH
authors.lastname: Baek
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Darlington
authors.forename: Paul F
authors.initials: PF
authors.lastname: Smith
authors.forename: John C
authors.initials: JC
authors.lastname: Ashton
mesh_terms: D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry / Q000379:methods / Q000592:standards*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D043885:Receptor, Cannabinoid, CB2 / Q000032:analysis*
chemical_list: D000906:Antibodies; D043885:Receptor, Cannabinoid, CB2