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Originally published as JHC exPRESS on January 9, 2006.
doi:10.1369/jhc.5A6827.2006
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Journal of Histochemistry and Cytochemistry
Volume 54 (5): 567-574, 2006
Copyright ©The Histochemical Society, Inc.

Generation and Characterization of Monoclonal Antibodies against the Transcription Factor Nkx6.1

Inger L. Pedersen, Rasmus Klinck, Jacob Hecksher-Sorensen, Stefan Zahn, Ole D. Madsen, Palle Serup and Mette C. Jorgensen

Antibody Core, Beta Cell Biology Consortium, www.betacell.org (ILP,ODM); Novo Nordisk A/S, Department of Antibody & Cell Technology, Bagsværd, Denmark (ILP,SZ); and Hagedorn Research Institute, Gentofte, Denmark (RK,JH-S,ODM,PS,MCJ)

Correspondence to: Dr. Palle Serup, Hagedorn Research Institute, Niels Steensensvej 6, DK-2820 Gentofte, Denmark. E-mail: pas{at}hagedorn.dk

We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine ß-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing ß-cells, making this transcription factor a unique ß-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/{kappa} subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the ß-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies. (J Histochem Cytochem 54:567–574, 2006)

Key Words: Nkx6.1 • pancreas • neural tube • development • diabetes • antibody


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