Generation and Characterization of Ptf1a Antiserum and Localization of Ptf1a in Relation to Nkx6.1 and Pdx1 During the Earliest Stages of Mouse Pancreas Development
J. Histochem. Cytochem. Hald et al. 56: 587

Supplemental Material - Hald et al. - Vol. 56, pp 587-595

Online Supplemental data for Wang et al.: Supplemental Figure, Supplemental Data, and Supplemental Movies (AVI).

Files in this Data Supplement:

  • Supplemental Data SD1 - Mouse, rat and human Ptf1a amino acid sequence are very similar over long stretches. Clustal W alignment of mouse, rat and human Ptf1a. Highlighted in green is the mouse GST-Ptf1a aa11-237 and in yellow are amino acids that are different from mouse. "*" means that the residues in that column are identical in all sequences in the alignment. ":" means that conserved substitutions have been observed. "." means that semi-conserved substitutions are observed.
  • Supplemental Figure SF1 - Further characterization of the Ptf1a anti-serum and control experiments. A: Western blot performed as in Figure 1. loaded with whole cell lysate of e15.5 pancreas. In lane 1 a double band around 45 kDa (marked by arrowheads) is observed when using rabbit anti-Nkx6.1 antibody as previously described (Jensen et al. 1996). Calculated molecular weight for Nkx6.1 is 37.7 kDa. By pre-incubating anti-Nkx6.1 with GST-Nkx6.1 the double band cannot be detected, lane 2, showing that the GST-Nkx6.1 used in Figure 1 lane 3 is functional. B: Cryosection of adult mouse pancreas stained for Ptf1a and amylase. Without antigen retrieval Ptf1a is observed in scattered cells from time to time (broken line marks an Islet). Compare to Figure 2A. C: Ptf1a staining of mouse e18.5 paraffin section. By pre-treatment with TEG buffer and abcam8212 a robust signal was observed on paraffin sections (broken line mark the border between endocrine and exocrine cells). D, E: Ptf1a antiserum tested on e15.5 mouse serial frozen sections. Similar nuclear Ptf1a staining is observed with Ptf1a antibody that had never been frozen (D) and Ptf1a antibody that had been frozen for 7 days at -20 oC (E). F: A triple staining for Ptf1a, amylase and Nkx6.1 on e15.5 mouse frozen sections shows that amylase and Ptf1a immunoreactivity co-localize to the same cells and that neither Ptf1a nor amylase positive cells co-stain for Nkx6.1. i: Islet, endo: endocrine, exo: exocrine. Scale bar is 50μm. Magnification is the same in C, F and B, D and E.
  • Supplemental Movie SM1A - Movie files of the entire confocal scans (A-J) corresponding to the Figure 3A-J horizontal panels. A-J: Throughout all panels Nkx6.1 is shown is green, Pf1a in red and Pdx1 in grey. The data shown in the movie files (SM1 A-J) and in Figure 3(A-J) originate from the same confocal scans. In Figure 3 only one or a merge of few optical sections are shown whereas all consecutive optical sections are available in the corresponding movie file. The indicated value in µm on each optical section indicate the distance from the first optical section.
  • Supplemental Movie SM1B - See Supplemental Movie SM1A.
  • Supplemental Movie SM1C - See Supplemental Movie SM1A.
  • Supplemental Movie SM1D - See Supplemental Movie SM1A.
  • Supplemental Movie SM1E - See Supplemental Movie SM1A.
  • Supplemental Movie SM1F - See Supplemental Movie SM1A.
  • Supplemental Movie SM1G - See Supplemental Movie SM1A.
  • Supplemental Movie SM1H - See Supplemental Movie SM1A.
  • Supplemental Movie SM1I - See Supplemental Movie SM1A.
  • Supplemental Movie SM1J - See Supplemental Movie SM1A.