Figure 4.

Immunodetection of IGF2R in organ cultures of normal or trephine-wounded porcine corneas. Untreated porcine corneas (A, B, F, H, J) or those wounded by removal of 5 mm trephined portion of the cornea, including epithelium and the anterior stroma (C, D, G, I, K), were mounted on a base containing agarose and collagen as described.⁴⁰ The corneas were cultured for 2 weeks, fixed, and sections were obtained. (A–D) Sections were incubated with IGF2R-specific polyclonal antibody (A, C) or the corresponding preimmune serum (B, D) followed by HRP-conjugated goat anti-rabbit antibody and colorimetric DAB substrate. Sections were counterstained with hematoxylin. A representative experiment is shown of three independent replicates. Arrows in (C) indicate regions of the stroma with intense staining for IGF2R. Quantification of IGF2R staining area in the control and wounded samples is shown in (E), and the values represent the mean ± SE. A 1-tailed Student's t-test, P < 0.001. (F–K) Immunofluorescence imaging was performed by double-labeling with antibodies specific for IGF2R ([F, G], green, Alexa Fluor488) and α-SMA ([H, I], red, Cy5), and nuclei are stained with DAPI (blue). Overlay of images is shown in (J, K). A representative experiment is shown of three independent replicates. Arrows in (G, I, K) highlight representative myofibroblasts that stain positive for both IGF2R and α-SMA. Scale bar: 100 μm.