Validation of a selective serotonin 5-HT_2C receptor antibody for utilization in fluorescence immunohistochemistry studies

Although radioligand binding studies have shown that the serotonin 5-HT_2C receptor (5-HT_2CR) is widely expressed throughout the brain, more detailed knowledge of 5-HT_2CR 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-HT_2CR 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-HT_2CR antibodies demonstrated that IR for the two antibodies largely overlapped. However, SC 5-HT_2CR IR was more concentrated within IR cell bodies and was more consistent among assays than the PH 5-HT_2CR IR. Thus, the SC 5-HT_2CR antibody was chosen for subsequent studies. When examined in 5-HT_2CR knockout vs. wild-type mice, the SC 5-HT _2CR antibody produced widespread IR in wild-type, but not 5-HT _2CR knockout, mice. In addition, 5-HT_2CR-IR was not present in either native CHO cells, known to be devoid of 5-HT_2AR or 5-HT_2CR, or in CHO cells transfected with the 5-HT_2AR. Thus, these studies suggest that the SC 5-HT_2CR antibody produces reliable staining selective for 5-HT_2CR vs. 5-HT_2AR in rodent brains and is therefore suitable for use in future immunofluorescence 5-HT_2CR localization studies.