Figure 6.

Confocal Microscopy Analysis of Aptamer H02 on U87MG α5+

(A) Confocal microscopy analysis with aptamer H02 at 5 μM and the anti-α5 antibody IIA1 on U87MG α5+ cells at 4°C and 37°C. The aptamer is labeled with Cy5 (white). Incubation of antibody IIA1 was followed by incubation with a secondary antibody labeled with Alexa 546 (green). Nuclei are stained with Hoechst (blue). (B) Top: enlargement of the merged image in (A) at 4°C. Bottom: magnified images acquired by zooming in on the indicated areas of the parental image. Scale bars are shown in the lower right corners of the images. (C) Co-localization of aptamer H02 and the endocytosis marker EEA1. Shown are confocal images of U87MG α5+ cells incubated at 37°C with the Cy5-labeled aptamer H02 at five different concentrations (5, 2.5, 1.25, 0.6, and 0.3 μM). After aptamer labeling (shown in white), cells were fixed, permeabilized, and then labeled to detect nuclei (DAPI, blue), actin (Phalloidin-Atto 488, green), and early endosome EEA1 (EEA1 immunolabeling, red). Shown in the first row are merged images. Shown in the second row are magnified images of selected areas (white squares) of the parental images. These images show co-labeling of EEA1 and aptamer H02. Shown in the third row are separate EEA1 and aptamer labeling. Images were captured at the same setting to allow direct comparison of staining patterns.