Validation of a selective serotonin 5-HT2C receptor antibody for utilization in fluorescence immunohistochemistry studies

Although radioligand binding studies have shown that the serotonin 5-HT2C receptor (5-HT2CR) is widely expressed throughout the brain, more detailed knowledge of 5-HT2CR distribution within different neuronal populations will aid in understanding the mechanisms through which this receptor acts. Double-label immunohistochemical procedures can be utilized to examine the localization of receptors within specific neuronal populations. In order to conduct such studies, however, it was first necessary to examine the utility and specificity of two commercially available anti-5-HT2CR antibodies [from Santa Cruz (SC) and BD PharMingen (PH)]. In male Sprague-Dawley rats, both antibodies produced widespread immunoreactivity (IR) throughout the brain area chosen for study, the ventral tegmental area, which is the origin of the dopamine mesocorticoaccumbens “reward” pathway. Co-labeling with the SC and PH 5-HT2CR antibodies demonstrated that IR for the two antibodies largely overlapped. However, SC 5-HT2CR IR was more concentrated within IR cell bodies and was more consistent among assays than the PH 5-HT2CR IR. Thus, the SC 5-HT2CR antibody was chosen for subsequent studies. When examined in 5-HT2CR knockout vs. wild-type mice, the SC 5-HT2CR antibody produced widespread IR in wild-type, but not 5-HT2CR knockout, mice. In addition, 5-HT2CR-IR was not present in either native CHO cells, known to be devoid of 5-HT2AR or 5-HT2CR, or in CHO cells transfected with the 5-HT2AR. Thus, these studies suggest that the SC 5-HT2CR antibody produces reliable staining selective for 5-HT2CR vs. 5-HT2AR in rodent brains and is therefore suitable for use in future immunofluorescence 5-HT2CR localization studies.