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. 2017 Jun 1;26(6):983–1000. doi: 10.3727/096368917X694697

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© 2017 Cognizant, LLC.

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Figure 4. — Colabeling of DH01 allografts with infiltrating CD11b⁺, F4/80⁺, and Gr1 Ly-6G⁺ cells peaks at POD 7 with evidence of graft engulfment by these innate immune cells. Large areas of SV40T were observed to colabel with CD11b⁺, F4/80⁺, and Gr1 Ly-6G⁺ on POD 7. Colabeling of SV40T with the T-lymphocyte marker (CD3-∊) remained low at all time points. Representative images of the immunofluorescence colabeling evident on POD 7 are shown. (A) The highlighted cell (white box) in the center of this image has positive cell membrane immunolabeling for the macrophage marker CD11b (TRITC, red) on POD 7. SV40T (FITC, green) immunolabeling can be seen within this CD11b⁺ cell, suggesting engulfment of graft by the macrophage. The differential interference contrast (DIC) image also identifies pigment granules within CD11b⁺ cells. Scale bar: 20 μm. (B) Multiple cells in this image have cell membrane immunolabeling for the macrophage marker F4/80 (TRITC, red) on POD 7. SV40T (FITC, green) immunolabeling can be seen within these F4/80⁺ cells. Areas of F4/80⁺/SV40T⁺ colabeling (orange) suggest graft engulfment by macrophages. The DIC image also identifies pigment granules within F4/80⁺ cells. Scale bar: 20 μm. (C) Many cells in this POD 7 subretinal graft have immunofluorescence colabeling (orange) for Gr1 Ly-6G (TRITC, red) and SV40T (FITC, green). Scale bar: 10 μm. (D) Sequential single optical sections from the z-stack confocal image through the highlighted (yellow box) Gr1 Ly-6G⁺ neutrophil on POD 7 is consistent with phagocytosis of SV40T from the neutrophil cell membrane. (E) The z-stack confocal image of the cell highlighted in (C) was also reconstructed in three-dimensional (3D) form, and the red channel was removed from the top portion of the cell to enable visualization inside the cell membrane. The reconstructed image confirms the presence of graft (SV40T, green) inside the neutrophil cell membrane (Gr1 Ly-6G, red). (F) The proportions of SV40T that colabeled with CD11b, F4/80, Gr1 Ly-6G, and CD3-∊ were analyzed. Colabeling of SV40T with all infiltrating immune cell markers was minimal on POD 1 and POD 3. However, there was a significant (^*p < 0.05) increase in the proportion of the graft (SV40T) that colabeled with F4/80, CD11b, and Gr1 Ly-6G between POD 1 and POD 7. Colabeling of SV40T with the T-lymphocyte marker (CD3-∊) remained low at all time points, and there was no statistically significant difference in SV40T/CD3-∊ colabeling between time points.