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 β-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing β-cells, making this transcription factor a unique β-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/κ subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the β-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)

The induction of pancreatic endocrine differentiation from undifferentiated precursor cells is a multi-step process regulated by the expression of several transcription factors. At E9.5 expression of homeodomain protein Pdx-1 defines the early pancreatic epithelium. Later, expression of the basic helix-loop-helix factor, Neurogenin 3 (Ngn3) marks the initiation of the endocrine lineage from the pancreatic precursor cells. A full understanding of these processes is essential in order to control development of insulin secreting beta-cells from embryonic stem cells in vitro. Since the expression of ngn3 is a key step, the aim of this work was to develop monoclonal antibodies for immunohistochemical (IHC) detection of Ngn3. All mice (RBF-strain) immunized with recombinant GST-mNGN3 fusion protein responded with a high antibody titer. The sera were further screened by IHC on paraffin sections of fetal (E13) mouse pancreas, and two hybridomas subsequently derived from one selected mouse produced antibodies with prominent and specific Ngn3 staining properties. These new antibodies provide novel useful tools for co-labeling studies using the large panel of existing rabbit polyclonal antibodies available against important transcription factors to further characterize the development of pancreatic endocrine progenitor cells.

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The aim of this study was to characterize two monoclonal antibodies (F6A11 and F109-D12) generated against Pdx1 (pancreatic and duodenal homeobox-1), a homeodomain transcription factor, which is critical for pancreas formation as well as for normal pancreatic beta cell function. For production of monoclonal antibodies, we immunized Robertsonian POSF (RBF)mice with a GST-Pdx1 fusion protein containing a 68-amino acid C-terminal fragment of rat Pdx1. These monoclonal antibodies detect Pdx1 by western blotting and allow immunohistochemical detection of Pdx1 in both mouse and rat tissue. F6A11 and F109-D12 produce IHC staining patterns indistinguishable from that obtained with highly specific polyclonal Pdx1 antisera raised in rabbits and goats, when applied to embryonic or adult mouse pancreatic tissue. In contrast to previously generated polyclonal anti-Pdx1 antisera, we also demonstrate that F6A11 works for intracellular fluorescence activated cell sorting (FACS) staining of Pdx1. By using F6A11, we characterize the induction of Pdx1 in the Doxycycline (DOX) inducible insulinoma cell line INSrαβ-Pdx1 and follow the reduction of Pdx1 after removing Dox. Finally, we show that induction of exogenous Pdx1 leads to a reduction in endogenous Pdx1 levels, which suggests that a negative feedback loop is involved in maintaining correct levels of Pdx1 in the cell.

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