Validation of a selective serotonin 5-HT(2C) receptor antibody for utilization in fluorescence immunohistochemistry studies

Abstract

Although radioligand binding studies have shown that the serotonin 5-HT(2C) receptor (5-HT(2C)R) is widely expressed throughout the brain, more detailed knowledge of 5-HT(2C)R 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(2C)R 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(2C)R antibodies demonstrated that IR for the two antibodies largely overlapped. However, SC 5-HT(2C)R IR was more concentrated within IR cell bodies and was more consistent among assays than the PH 5-HT(2C)R IR. Thus, the SC 5-HT(2C)R antibody was chosen for subsequent studies. When examined in 5-HT(2C)R knockout vs. wild-type mice, the SC 5-HT(2C)R antibody produced widespread IR in wild-type, but not 5-HT(2C)R knockout, mice. In addition, 5-HT(2C)R-IR was not present in either native CHO cells, known to be devoid of 5-HT(2A)R or 5-HT(2C)R, or in CHO cells transfected with the 5-HT(2A)R. Thus, these studies suggest that the SC 5-HT(2C)R antibody produces reliable staining selective for 5-HT(2C)R vs. 5-HT(2A)R in rodent brains and is therefore suitable for use in future immunofluorescence 5-HT(2C)R localization studies.