Development of a 5-HT7 receptor antibody for the rat: the good, the bad, and the ugly

• Published in: Naunyn-Schmiedeberg's archives of pharmacology Vol. 396; no. 10; pp. 2599 - 2611
• Main Authors: Thompson, Janice M., Tragge, Will, Flood, Emma D., Schulz, Stefan, Lisabeth, Erika, Watts, Stephanie W.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.10.2023; Springer Nature B.V
• Subjects: Antibodies; Antigens; Biomedical and Life Sciences; Biomedicine; Blood pressure; C-Terminus; Neurosciences; Pharmacology/Toxicology; Serotonin S7 receptors; Veins; Immunohistochemistry; Westerns; receptor; GPCR antibody; 5-HT
• ISSN: 0028-1298; 1432-1912; 1432-1912
• DOI: 10.1007/s00210-023-02482-w

Our laboratory has a vested interest in measuring the location and expression of the 5-hydroxytryptamine (5-HT, serotonin) 7 (5-HT 7 ) receptor in the rat. Determining tissue-specific receptor expression would aid in validating understood and potentially new tissues that support the 5-HT 7 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-HT 7 (r5-HT 7 ) 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-HT 7 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-HT 7 receptor [ERPERSEFVLQNSDH(Abu)GKKGHDT; antibodies 3, 6, and 9] positively and concentration-dependently identified the r5-HT 7 receptor expressed in Westerns of transfected HEK293T cells. These same C terminus antibodies also successfully detected the r5-HT 7 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-HT 7 receptor in rat veins. This deliberate work has given rise to at least three antibodies that can be used with good confidence in r5-HT 7 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.