Fig 2: H&E and immunofluorescent staining for tPA (green), cell nuclei are stained blue. Magnification × 20 and on day 10 × 40. ME, middle ear; EAM, external acoustic meatus; M, malleus handle; single asterisk indicates proliferating epithelium control. Pars tensa of TM, section through malleus handle (H&E). Immunofluorescent staining for tPA is visible in the thin layer of epithelium covering external surface of the TM mainly in the malleus region and in the superficial layers of epithelium covering external acoustic meatus. The green signal visible inside the EAM space and in the sebaceous glands presumably comes from sebum autofluorescence. Day 1, the area near the umbo with a layer of proliferating epithelium (H&E). Immunofluorescent staining for tPA visible in the proliferating epithelium. Day 2, section through the malleus handle with proliferating epithelium covering the surface of malleus handle and surrounding it on the side of perforation (H&E). Immunofluorescent staining for tPA visible in the proliferating epithelium. Day 3, section through the lower part of the malleus handle, the ridge of proliferating epithelial cells near the malleus, migrating on the external surface of TM remnant (H&E). Immunofluorescent staining for tPA visible in the proliferating epithelium. Day 5, section through the malleus handle with proliferating epithelium and thick layer of proliferating fibroblasts adjacent to the perforation (H&E). Immunofluorescent staining for tPA clearly visible in the layer of proliferating squamous epithelium. Day 7, section through the malleus handle with proliferating epithelium (H&E). Immunofluorescent staining for tPA visible in the proliferating epithelium. Day 10, closed perforation with uneven layer of squamous epithelium and loose connective tissue composed of fibroblasts (H&E). Immunofluorescent staining for tPA visible in the epithelium, especially in its superficial layer