Fig. S4

Single confocal slices of
histological sections from a 7 days post amputation (dpa) Tg(kdrl:mCherry-ras) heart double immunostained to detect Wif1 (A,A’; yellow) or Notum1b (B,B’; cyan) and the endocardial reporter mCherry (A,A’’,B,B’’; red). Merged (A,B) and single channel (A’,A’’,B’,B’’) images are shown. (C-G’’) Single confocal slices of histological sections from a 7 dpa heart stained with a pan- Cytokeratin antibody (PKC; red), costained with the myocardial specific MF20 antibody (green), and counter-stained with DAPI (blue). The lowest magnification images of the ventricle and atrium are shown in C-C’’. Higher magnification images of the ventricular and atrial walls are shown in (D) and (F), respectively. The boxed regions in (E) and (G) are shown at the highest magnification in (E-E’’) and (G-G’’), respectively. Single channel (C’,C’’) and merged double (C,E’,E’’,G’,G’’) or triple (D,E,F,G) channel images are shown. (H-K’’) Single confocal slices of histological sections from a 7 dpa heart stained with the Wif1 (H-I’’; yellow) or Notum1b (J-K’’; cyan) antibody, costained with the PCK antibody (deep pink) and counter-stained with DAPI (blue). Images of the wound region (H-H’’,J-J’’) and spared portion of the ventricle (I- I’’,K-K’’) are shown. Single channel (H’,H’’,J’,J’’) and merged double (H,J,I’,I’’,K’,K’’) or triple (I,K) channel images are shown. (L-M””) Single confocal slices of histological sections from a 7 dpa heart stained with the Wif1 (L-L””; yellow) or Notum1b (M-M””; cyan) antibody and costained with antibodies that recognize fibroblasts [Collagen, type I, alpha 1 (Col1a1) antibody; dark orange] or cardiomyocytes (MF20 antibody; magenta). Images of the wounded region (L-M””) are shown. Single channel (L”-L””; M”- M””) and merged double (L’,M’) or triple (L,M) channel images are shown. In all cases, greater than three sections from each of four hearts were examined. Little to no variability in protein localization was observed. Scale bars=50μm for (A-B’’,D-M””) and 200μm for (C-C’’).