The corneal tissue samples were obtained from experimental animals that received transplants of hIDPSC and of AM, were fixed with 4% paraformaldehyde (Sigma), and embedded (Tissue-Tek OCT [optimal cutting temperature]; Sakura, Torrance, CA). Frozen sections (5 μm) were obtained using a cryomicrotome (Cryostat CM1100; Leica, Wetzlar, Germany) and placed on poly-l-lysine-coated slides, which were incubated in cold methanol (Sigma) for 15 minutes to decrease tissue autofluorescence. Afterward, they were washed three times in rinse buffer (TBS) containing: 20 mM Tris-HCl (Vetec, Duque de Caxias, Brazil), pH 7.4; 0.15 M NaCl (Dinâmica Reagent, Sao Paulo, Brazil); and 0.05% Tween-20 (Sigma). Permeabilization was performed using 0.1% Triton X-100 (Santa Cruz Biotechnology) for 30 minutes. The slides were washed three times and incubated for 1 hour with 5% bovine serum albumin (BSA; Sigma) in PBS (Gibco), pH 7.4. Primary antibodies were diluted in PBS and the slides were incubated for 1 hour at indicated dilution: anti–β1-integrin, anti-CK18, anti-K3, anti-K12, anti-vimentin, anti-ABCG2 (1:100), anti-p63 (1:200), and anti-hIDPSC (1:500) at room temperature. After washing in TBS (three times), the slides were incubated in the dark for 1 hour with secondary anti-mouse antibody conjugated with fluorescein isothiocyanate (FITC) or cianine dye 3 (Cy3) both at a dilution of 1:500. The same concentration of corresponding normal non-specific IgG provided the negative control. Frozen sections obtained from rabbits that received only AM and native rabbit corneal tissue were also used as controls. After washing with PBS, microscope slides were mounted in antifade solution (Vectashield mounting medium; Vector Laboratories, Hercules, CA) with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI) and analyzed by fluorescent microscopy (Axio Imager A1; Carl Zeiss) or confocal microscopy (LSM 510 META; Carl Zeiss). An argon ion laser set at 488 nm for FITC (Chemicon) and at 536 nm for Cy3 (Chemicon) excitation was used. The emitted light was filtered with a 505 nm (FITC) and 617 nm (Cy3) long pass filter in a laser scanning microscope.