Antibody testing for brain immunohistochemistry: Brain immunolabeling for the cannabinoid CB2 receptor

•Three antibodies for the CB2 receptor, which have been previously used to describe immunolabeling in the uninjured brain, were tested.•None of the antibodies were confirmed using the knockout control test.•Confirmation of these antibodies using other tests indicated that those tests cannot be used to validate the antibodies.•Claims of widespread CB2 expression in neurons in the uninjured brain are thereby cast into doubt. 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.