HIMF and Stem Cells Figure 5. GFP and cellular markers sca-1 and c-kit co-localize in AAV-HIMF-treated bone marrow transplant recipients. Frozen lung sections from bone marrow transplant recipients treated with AAV-HIMF (2.561010 VP, 14d) were stained with antibodies for (B) sca-1, (F) c-kit, or (J) CD34 (red). (C, G, K) GFP signal was obtained through direct visualization (green). (A, E, I) Cell nuclei were counterstained with DAPI (blue). (D, H, L) The arrows in the merged images demonstrate co-localization of GFP with the cellular markers. Scale bar: 20 mm. doi:10.1371/journal.pone.0011251.g005 Figure 6. GFP+ cells were recruited to the smooth muscle layer of the pulmonary vasculature in AAV-HIMF-treated mice. GFP was detected through direct visualization (A, E; green). HIMF and a-smooth muscle actin (a-SMA) were detected with anti-HIMF and anti-a-SMA primary antibodies and visualized with rhodamine-conjugated anti-rabbit IgG secondary antibodies (B; red) and Cy5-conjugated anti-mouse IgG secondary antibodies (C; pink), respectively. Arrows in the merged image indicate co-localization of GFP, HIMF, and a-SMA (D). Lung sections were stained with anti-CD31 antibodies and visualized with rhodamine-conjugated anti-rat IgG antibodies (F; red) and anti-a-SMA antibodies and visualized with Cy5-conjugated anti-mouse IgG antibodies (G, pink). (F) Arrows in the merged image indicate co-localization of GFP and a-SMA. Cell nuclei were stained with DAPI (D, H; blue) Scale bar: 20 mm. doi:10.1371/journal.pone.0011251.g006 PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11251