Our laboratory has a vested interest in measuring the location and expression of the 5-hydroxytryptamine (5-HT, serotonin) 7 (5-HT7) receptor in the rat. Determining tissue-specific receptor expression would aid in validating understood and potentially new tissues that support the 5-HT7 receptor-mediated fall in blood pressure, an event we are committed to understand. We contracted with 7TM Antibodies to develop deliberately and rigorously a rat 5-HT7 (r5-HT7) receptor specific antibody. Three antigens, two targeting the third internal loop and one the C terminus, were used in three rabbits to generate antibodies. As a positive control, HEK293(T or AD) cells were transfected with a plasmid for the r5-HT7 receptor also expressing a C terminus 3xFLAG tag. Naïve rat tissues were also used in Western and immunohistochemical analyses. Nine antibodies (3 from three different rabbits) detected a ~ 75 kDa protein absent in homogenates of vector control HEK293T cells. Only antibodies that recognized the C terminus of the 5-HT7 receptor [ERPERSEFVLQNSDH(Abu)GKKGHDT; antibodies 3, 6, and 9] positively and concentration-dependently identified the r5-HT7 receptor expressed in Westerns of transfected HEK293T cells. These same C terminus antibodies also successfully detected the r5-HT7 receptor in immunocytochemical test of the transfected HEK293AD cells, colocalizing with the detected FLAG sequence. In naive tissue, antibody 6 performed the best, identifying specific bands in the brain cortex in Western analysis. These same antibodies produced a more diverse band profile in the vena cava, identifying 6 major proteins. In immunohistochemical experiments, the same C-terminus antibodies, with antibody 3 performing the best, detected the 5-HT7 receptor in rat veins. This deliberate work has given rise to at least three antibodies that can be used with good confidence in r5-HT7 transfected cells, two antibodies that can be used in immunohistochemical analyses of rat tissues and in Westerns of rat brain; we are less confident of the use of these same antibodies in rat veins.

Our laboratory has a vested interest in measuring the location and expression of the 5‐hydroxytryptamine (5‐HT, serotonin) 7 (5‐HT7) receptor. The 5‐HT7 receptor is responsible for the fall in blood pressure when 5‐HT is infused acutely or chronically in the rat. Determining tissue‐specific receptor expression would aid in understanding mechanisms that support the 5‐HT7 receptor‐mediated fall in blood pressure. We have, using tissues from a 5‐HT7 receptor KO rat we created, tested over a dozen commercially available 5‐HT7 receptor antibodies. None can discriminate between WT and KO tissues, indicating a lack of specificity that has been found commonly with antibodies directed against G protein coupled receptors (GPCRs), of which the 5‐HT7 receptor is one. We contracted with 7TM antibodies (Germany) to develop rigorously a 5‐HT7 receptor specific antibody, knowing there was a 50% chance of failure. Three different antigens, two targeting the third internal loop and one the C terminus, were used in rabbits to generate antibodies. We sent 7TM a plasmid for the r5‐HT7 receptor which, transiently expressed in HEK293 cells, served as a positive control for screening initial antibodies. In Western analyses done in Germany, nine (9) antibodies (all antigens represented) detected a ~75 kDa protein that was not present in homogenates of non‐transfected HEK293 cells. These same antibodies were sent to MSU for testing in the following ways: analyses of r5‐HT7 receptor expressing HEK293 cells to demonstrate protein dependence of antibody binding in Westerns; immunohistochemical analyses of brains from 5‐HT7 receptor WT and KO rats. Only antibodies that recognized the C terminus of the 5‐HT₇ receptor [ERPERSEFVLQNSDH(Abu)GKKGHDT] positively and concentration‐dependently identified the r5‐HT7 receptor expressing HEK293 cells in Westerns. By contrast, these same C‐terminus antibodies have not specifically detected the 5‐HT7receptor in rat tissues. No specificity of signal was observed between brain sections of WT and KO rats when tested immunohistochemically. Future studies will explore different protein isolation and immunoprecipitation in buffers specialized for GPCR extraction with the goal of increased specificity of antibody signal.