Dc 200

Bright-field images of the AM-stained preparations were acquired by using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) and a high resolution digital camera (DC 200, Leica Microsystems). At a primary magnification of ×20 one field per section, randomly chosen within the CB tissue, was selected and its image acquired in full colors (RGB, 24-bit), processed to correct shading, then filed TIFF (Figure 1A).
Since it showed the best contrast between the connective tissue and the CB parenchima, the red component of each acquired RGB image was selected for further processing (Figure 1B). Stromal structures and filaments can be easily segmented with conventional thresholding methods, and small remaining artifacts can be removed from the resulting binary image by applying a geometric filter to eliminate profiles within a specified range of area and/or shape (see Russ, 2011 ), leading to the generation of binary images (Figure 1C) of the connective component.
The amount of CB tissue it accounts for can be directly estimated from the corresponding binary image by evaluating the area fraction occupied by the binary pattern (Russ and Dehoff, 2000 ).

In corneal epithelium apoptotic cells were detected using a commercially available fluorescence kit (ApopTag Plus Fluorescein in situ Apoptosis Detection Kit, Chemicon International, Temecula, CA, USA) based on the TUNEL method, which detects and labels the free 3′-OH end of DNA strand breaks in apoptotic nuclei. According to the manufacturer's protocol, sections were fixed in 1% PFA solution after washing with PBS. Digoxigenin-labeled nucleotides in reaction buffer and terminal deoxynucleotidyl transferase enzyme (TdT) were applied to the sections for one hour at 37°C to catalyze the template-independent addition of nucleotide triphosphates to the 3′-OH ends of double-stranded or single-stranded DNA. After termination of the reaction, fluorescent-labeled antidigoxigenin antibodies were applied to visualize the nucleotides added to DNA free ends. Sections were counterstained with DAPI and visualized using fluorescence microscopy [22 , 23 ]. Microscopic analysis was performed with a Leica DMLB microscope equipped with epifluorescence EL6000 system (Leica Microsystems, Solms, Germany). Images were captured with a CCD camera (DC 200, Leica Microsystems, Solms, Germany) and image analysis software Quantimet 520 (Leica Microsystems, Solms, Germany). The number of apoptotic nuclei that stained intensely green was expressed relative to total number of nuclei stained by DAPI.