Three severe pterygium tissue samples (grade T3) were obtained intra-operatively. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Briefly, all paraffin sections (4 μm) were deparaffinized in xylene and rehydrated, and endogenous peroxidase was quenched. Cryostat sections were placed on gelatinized slides and fixed in cold acetone. Tissue sections were equilibrated in tris-buffered saline (TBS), blocked in nonimmune serum (Zymed Laboratories, South San Francisco, CA), and incubated with monoclonal mouse antibodies against human SDF-1 (1:2000; Abcam, Inc., Cambridge, MA) or CXCR4 (1:2000; Abcam, Inc.) overnight at 4°C. Sections were washed in TBS before adding biotinylated secondary antibody, washed again, incubated for 1 hour with peroxidase-conjugated streptavidin, and then the presence of peroxidase was revealed by adding substrate-chromogen (3-amino-9-ethycarbazole) solution. The sections were then counterstained with hematoxylin, examined under an optical microscope (Axioskop 40; Carl Zeiss, Göttingen, Germany), and photodocumented.