Specific staining of nuclear Ptf1a-like immunoreactivity was completely abolished by preabsorption to GSTCPtf1a (the fusion protein used as immunogen, Figure 2F compared with Figure 2E). antibody raised against the c-terminal portion of the mouse Ptf1a protein and report immunodetection, for the first time, as early as embryonic day (e) 8.5Ce8.75 in the dorsal and ventral buds of the mouse pancreas as well as Mitoquinone mesylate in the neural tube at e10.0. Detailed confocal analysis identifies an abundant triple-positive (Ptf1a+/Nkx6.1+/Pdx1+) putative early multipotent pancreatic progenitor cell that marks the e9.5 dorsal pancreas and e10.5 ventral pancreas. Furthermore, expression patterns of Nkx6.1 vs Ptf1a subsequently segregate during branching morphogenesis (trunk vs tip), ending up marking two distinct cell populations of progenitors at e12.5. From e15.5 (mouse) and in adult pancreas (mouse, rat, and human), the Ptf1a antibody marks only acinar cell nuclei, as expected for its subsequent role in committing/maintaining cells in this differentiated state. In summary, this antibody is a novel tool to further characterize important early steps of pancreas differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:587C595, 2008) Keywords: Ptf1a, p48, pancreas, whole mount, antibody, Nkx6.1, Pdx1, progenitor, stem cell Pancreas transcription factor 1a (Ptf1a), also known as p48, was first described in 1989 (Roux et al. 1989) as a basic helixCloopChelix (bHLH) transcription factor that is part of the trimeric PTF1 complex. Using RT-PCR, Ptf1a mRNA was reported as detectable from embryonic day (e) 12 and by in situ hybridization (ISH) at e14 in the just-forming acinar cells (Krapp et al. 1996). In a later study, Ptf1a mRNA expression was already detected in the early pancreatic buds at e9.5C10.0 by ISH, as well as in a thin stripe of the dorsal part of the neural tube (Obata et al. 2001). The first global deletion of Ptf1a resulted in an apancreatic phenotype with endocrine pancreatic cells reported in the spleen (Krapp et al. 1998). Lineage tracing studies using pPtf1a CRE/R26R mice allowed detection of Ptf1a+ cells in the dorsal and ventral pancreas beginning at e10.0Ce10.5 and, more importantly, demonstrated that Ptf1a is expressed in the multipotent pancreatic progenitors giving rise to both exocrine and endocrine pancreatic cells (Kawaguchi et al. 2002). Moreover, this latter Ptf1a ablation study was able Rabbit Polyclonal to Collagen XI alpha2 to follow the progeny of Ptf1a-deficient cells and showed that although a small rudimentary pancreatic outgrowth formed from the dorsal pancreas bud, most of the Ptf1a-deficient progeny of the dorsal and ventral buds converted into duodenum (Kawaguchi et al. 2002). Ptf1a immunoreactivity has been localized to the nucleus of acinar Mitoquinone mesylate cells of the adult mouse pancreas (Beres et al. 2006) Mitoquinone mesylate using a polyclonal rabbit antibody raised Mitoquinone mesylate against a synthetic amino acid (aa) peptide corresponding to the carboxyl-terminal 16 aa of mouse and rat Ptf1a (Rose et al. 2001). A rabbit antibody generated against a glutathione-S-transferase (GST)CPtf1a (mouse) fusion protein gave prominent nuclear staining of most cells at e10.5 in the dorsal and ventral pancreas (Li and Edlund 2001; J?rgensen et al. 2007) as well as nuclear staining of adult acinar cells (Hart et al. 2003). Real’s group also reported an affinity-purified polyclonal rabbit antibody for immunohistochemical application (Adell et al. 2000). Despite these published antibodies, it has remained difficult to detect with consistency and specificity the Ptf1a protein during its first phase of expression (Zhou et al. 2007). We now describe a novel rabbit antibody raised against GSTCPtf1a (mouse aa 11C237) with which we can robustly detect Ptf1a immunoreactivity as early as day e8.75 Mitoquinone mesylate both in ventral and dorsal pancreatic buds of mouse. We also report specific antigen retrieval and immunodetection conditions in which robust signals can be obtained in both human and rodent tissues to facilitate detection of this protein by other members of the community. The antibody has been produced in a substantial quantity and affinity that will serve as an important tool to further characterize the early multipotent pancreatic progenitor cells and potentially be useful in defining specific qualities of cells generated by induction programs in differentiating embryonic stem (ES) cells in vitro..