Fig 1: H&E and immunofluorescent staining for uPA (red), uPAR (pink), and PAI-1 (yellow), cell nuclei are stained blue. Magnification × 20 and × 40 on day 10. ME, middle ear; EAM, external acoustic meatus; A, tympanic annulus; single asterisk indicates proliferating epithelium control. Pars tensa of the TM, area around the annulus (H&E). Immunofluorescent staining visible in the thin layer of epithelium covering external surface of TM, mainly in the annular region, and in the epithelium covering neighboring meatus. Staining is also visible in the outer portion of annulus, especially prominent for uPA, and weak for uPAR and PAI-1. Day 1, the area near the annuls with proliferating epithelium, migrating on the surface of TM remnant (H&E). Immunofluorescent staining visible in the proliferating epithelium. The green signal visible on PAI-1 stain presumably comes from sebum autofluorescence. Day 2, the same area as on day 1 with thicker layer of proliferating epithelium migrating on TM remnant (H&E). Immunofluorescent staining visible in the proliferating epithelium. The green signal visible on PAI-1 stain presumably comes from sebum autofluorescence. Day 3, thick layer of proliferating epithelium on the external surface and thinner on the inner surface of TM in the vicinity of annulus (H&E). Immunofluorescent staining visible in the proliferating epithelium on the external surface of TM and in the superficial layer of proliferating epithelium on the inner side. Day 5, the edge of perforation and remnant of fibrous layer of pars tensa surrounded by proliferating squamous epithelium (H&E). Immunofluorescent staining prominent in the layer of proliferating squamous epithelium especially close to the growing front. Weak staining for uPA and uPAR is also present in proliferating fibroblasts. Day 7, the edge of perforation and remnant of fibrous layer of pars tensa surrounded by proliferating fibroblasts and thinner than on day 5 proliferating squamous epithelium on external side of TM. Keratin squames with inflammatory cells on its front (H&E). Immunofluorescent staining visible in the layer of proliferating squamous epithelium and in the thin mucosal layer. Day 10, closed perforation with the layer of squamous epithelium consisting of several layers and loose connective tissue composed of fibroblasts (H&E). Immunofluorescent staining clearly visible in the epithelial layer