Axiophot hbo 50

After transmittance measurements, the corneas were fixed with 4% buffered paraformaldehyde and embedded in paraffin. Sections 5-μm thick were stained with Picrosirius-red (0.1% Sirius red in saturated aqueous picric acid for 1 h) (Junqueira et al., 1979) . Sections were examined under an Axiophot microscope (Zeiss Axiophot HBO-50; Carl Zeiss, Oberkochen, Germany) with polarized light. Photomicrographs were captured using the AxioCam HRc Digital Camera and Axiovision release 4.8 software (Carl Zeiss) under the same plane of polarization. Polarization colors are associated with different collagen fiber thicknesses, packing density, and spatial arrangement (Dayan et al., 1989) . Orange to red colors were correlated with thick collagen fibers that were tightly packed and well aligned. Yellowish-green colors were correlated with thinner fibers that were less tightly packed.
Other 5-μm sections were stained with a modification of the Gomori's Silver Impregnation technique to demonstrate the presence of collagen type III fibers [54] . The sections were examined under an Axiophot microscope (Zeiss Axiophot HBO-50; Carl Zeiss, Oberkochen, Germany) and photomicrographs were obtained with an AxioCam HRc Digital Camera and Axiovision release 4.8 software (Carl Zeiss).

Sections 5-μm thick were deparaffinized and alpha-smooth muscle actin (αSMA) presence, a marker of myofibroblasts, was identified by incubation with mouse monoclonal anti-human αSMA Clone 1A4 Ready-to-use (Dako, Glostrup, Denmark). The secondary antibody was FITC goat anti-mouse IgG (Molecular Probes). Nuclei were stained with DAPI (Molecular Probes). Immunofluorescence sections were examined under an Axiophot fluorescence-incorporated microscope (Zeiss Axiophot HBO-50; Carl Zeiss, Oberkochen, Germany) and photomicrographs were captured using the AxioCam HRc Digital Camera and Axiovision release 4.8 software (Carl Zeiss). All the αSMA-positive cells were then counted at 100X magnification using the Touch Count function from Cell A software (Olympus) in 300,000 μm 2 areas of the wound and peripheral area. The percentages were determined relative to the total cell number at each time point.
All images were taken under the same conditions and experiments were performed at least four times. Limbal blood vessels were used as