LETTER TO THE EDITOR Cannabinoid CB2 receptor immunolabelling in the healthy brain—still a live possibility John C. Ashton & Yiwen Zheng & Cynthia Darlington & Jean-Ha Baek & Paul F. Smith Received: 21 November 2013 /Accepted: 5 December 2013 /Published online: 18 December 2013 # Springer-Verlag Berlin Heidelberg 2013 In their comprehensive and rigorous study, Cécyre and col- leagues (Cécyre et al. 2013) were able to demonstrate cannabinoid CB2 receptor-specific labelling for a Cayman Chemical polyclonal antibody in the mouse retina. This con- trasts with our (Baek et al. 2013) result using the same antibody for the mouse brain . The authors suggest that this discrepancy may be a result of the greater number of possible cross-reacting proteins in the brain compared with the retina. We would like to take the opportunity to put forward an alternative hypothesis, one that is perhaps more optimistic with respect to the use of this antibody on brain tissue. First, we note that the same antibody appears to bind to a cross- reacting protein in the retina when Western blot is employed (Cécyre et al. 2013). Therefore, there is at least one other protein in the retina with an epitope that can bind to the antibody if the epitope is sufficiently accessible. Therefore, it appears that there is a protein with such an epitope in the retina such that it is available for antibody binding under the conditions of Western blot but not immunochistochemistry in the Cécyre et al. (Cécyre et al. 2013) study. Hence, we propose that the tissue processing procedures employed in their exper- iments were sufficient to unmask the epitope for CB2, whilst leaving the epitope for a potentially cross-reacting protein inaccessible to the antibody. By contrast, it could be that the procedures that we (Baek et al. 2013) employed in our own study unmasked both epitopes. If this hypothesis is correct, then it leaves the question of CB2 immunolabelling in the brain open. Hence, it would be very interesting if the protocols of Cécyre et al. (Cécyre et al. 2013) were employed for mouse WT and CB2 KO brain tissue—as if CB2 is present in the healthy brain, then such a procedure might be able to detect it. Conflict of interest The authors have no conflicts of interest to declare. References Baek JH, Darlington CL, Smith PF, Ashton JC (2013) Antibody testing for brain immunohistochemistry: brain immunolabeling for the cannabinoid CB (2) receptor. J Neurosci Methods 216:87–95 Cécyre B, Thomas S, Ptito M, Casanova C, Bouchard JF (2013) Evaluation of the specificity of antibodies raised against cannabinoid receptor type 2 in the mouse retina. Naunyn-Schmiedeberg's Arch Pharmacol J. C. Ashton (*) : Y. Zheng : C. Darlington : J.<H. Baek : P. F. Smith Department of Pharmacology and Toxicology, School of Medical Sciences, University of Otago, P.O. Box 913, Dunedin, New Zealand e-mail: [email protected] Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:301 DOI 10.1007/s00210-013-0948-y

Cannabinoid CB2 receptor immunolabelling in the healthy brain—still a live possibility

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LETTER TO THE EDITOR

Cannabinoid CB2 receptor immunolabelling in the healthybrain—still a live possibility

John C. Ashton & Yiwen Zheng & Cynthia Darlington &

Jean-Ha Baek & Paul F. Smith

Received: 21 November 2013 /Accepted: 5 December 2013 /Published online: 18 December 2013# Springer-Verlag Berlin Heidelberg 2013

In their comprehensive and rigorous study, Cécyre and col-leagues (Cécyre et al. 2013) were able to demonstrate canna-binoid CB2 receptor-specific labelling for a CaymanChemical polyclonal antibody in the mouse retina. This con-trasts with our (Baek et al. 2013) result using the same anti-body for the mouse brain . The authors suggest that thisdiscrepancy may be a result of the greater number of possiblecross-reacting proteins in the brain compared with the retina.We would like to take the opportunity to put forward analternative hypothesis, one that is perhaps more optimisticwith respect to the use of this antibody on brain tissue. First,we note that the same antibody appears to bind to a cross-reacting protein in the retina when Western blot is employed(Cécyre et al. 2013). Therefore, there is at least one otherprotein in the retina with an epitope that can bind to theantibody if the epitope is sufficiently accessible. Therefore,it appears that there is a protein with such an epitope in theretina such that it is available for antibody binding under theconditions of Western blot but not immunochistochemistry inthe Cécyre et al. (Cécyre et al. 2013) study. Hence, we proposethat the tissue processing procedures employed in their exper-iments were sufficient to unmask the epitope for CB2, whilst

leaving the epitope for a potentially cross-reacting proteininaccessible to the antibody. By contrast, it could be that theprocedures that we (Baek et al. 2013) employed in our ownstudy unmasked both epitopes. If this hypothesis is correct,then it leaves the question of CB2 immunolabelling in thebrain open. Hence, it would be very interesting if the protocolsof Cécyre et al. (Cécyre et al. 2013) were employed for mouseWT and CB2 KO brain tissue—as if CB2 is present in thehealthy brain, then such a procedure might be able to detect it.

Conflict of interest The authors have no conflicts of interest to declare.

References

Baek JH, Darlington CL, Smith PF, Ashton JC (2013) Antibody testingfor brain immunohistochemistry: brain immunolabeling for the can-nabinoid CB (2) receptor. J Neurosci Methods 216:87–95

Cécyre B, Thomas S, Ptito M, Casanova C, Bouchard JF (2013)Evaluation of the specificity of antibodies raised against cannabi-noid receptor type 2 in the mouse retina. Naunyn-Schmiedeberg'sArch Pharmacol

J. C. Ashton (*) :Y. Zheng : C. Darlington : J.<H. Baek :P. F. SmithDepartment of Pharmacology and Toxicology, School of MedicalSciences, University of Otago, P.O. Box 913, Dunedin, New Zealande-mail: [email protected]

Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:301DOI 10.1007/s00210-013-0948-y