Fig 2: Retinal flat mounts from T1D (A–F) human retinas, double stained with CD39 (green) and IDO (red) or QUIN (red). A, B, and C and D, E, and F show the same fields of view. A and D only show CD39 labeling. B only shows IDO labeling. E shows only QUIN labeling. C is the merged image of A and B. F is the merged image of D and E. The images A–C show some CD39+ microglia (arrow in A) were IDO positive (arrows in B and C). The images D–F (higher magnification from Figs. 4C, 4D) show some CD39+ microglia are QUIN positive. Calibration in A: for A–F.

Fig 3: Retinal flat mounts from nondiabetic (A, B) and T1D (C, D) rat retinas, double stained with IDO (red) and GS lectin (green). A and B, C and D are the same images, with A and C only showing IDO labeling. The images in A and B show weak IDO+ expression on GS lectin+ microglia and GS lectin+ blood vessels. In T1D (C, D), IDO+/Lectin+ microglia (arrows) increased in density and brightness, and IDO+ labeling on blood vessels also increased when compared with control. (E) Quantitative analysis shows IDO+ microglia-like cell density was significantly higher in T1D (2.76 times; *P < 0.05) retinas when compared with nondiabetic controls. N = 3 samples/group. (F) Quantitative analysis shows IDO+ cell brightness (intensity) was significantly higher in T1D (2.06 times; *P < 0.05) retinas when compared with nondiabetic controls. (G) Quantitative analysis shows that the bright IDO+ expression on BVECs was significantly higher in T1D (1.88 times; *P < 0.05) retinas when compared with nondiabetic controls. Calibration in A: for A–D.

Fig 4: Retinal flat mounts from nondiabetic (A, B), T1D (C, D), and T2D (E, F) human retinas, triple stained with IDO (red), UEA lectin (green), and CD39 (blue). A and B, C and D, E and F are the same images, with A, C, and E showing only IDO labelling. The images in A and B show weak IDO+ expression on CD39+ microglia and CD39+/UEA lectin+ blood vessels. In T1D and T2D, IDO+ microglia increased in density and brightness, but IDO expression on blood vessels did not change. (G) Quantitative analysis shows IDO+ microglia-like cell density was significantly higher in both T1D (53%) and T2D (56%; *P < 0.05) retinas when compared with nondiabetic controls. N = 4 to 6 samples/group, and there was no difference between T1D and T2D. (H) Quantitative analysis shows IDO+ cell brightness (intensity) was significantly higher both in T1D (68%) and T2D (54%; *P < 0.05) retinas when compared with nondiabetic controls. N = 4 to 6 samples/group, and there was no difference between T1D and T2D. (I) Quantitative analysis shows that the bright IDO+ expression on blood vessel endothelial cells (BVECs) was similar among the T1D, T2D, and nondiabetic control groups. (N = 4–6 samples/group). Calibration in A: for A–F.