To investigate and differentiate the expression pattern of ANG in pterygia and normal conjunctiva, 6 pterygial tissue samples, including 3 specimens with all of four severe phenotypes (with recurrence, active form, grade T3, and grade V3), and 3 specimens with none of the severe phenotypes (primary, inactive form, grade T1, and grade V1), and an additional 2 normal conjunctiva samples were obtained among the enrolled patients for immunohistochemical (IHC) staining of ANG. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Briefly, all paraffin sections (4 μm) were deparaffinized in xylene, rehydrated, and quenched with endogenous peroxidase. 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 mouse monoclonal antibody against human ANG (1:500; Abcam, Inc., Cambridge, MA) overnight at 4°C. Sections were washed in TBS before adding the biotinylated secondary antibody, rewashed, and incubated for 1 hour with peroxidase-conjugated streptavidin, and the presence of peroxidase was revealed by adding substrate-chromogen (3-amino-9-ethycarbazole) solution. The sections then were counterstained with hematoxylin, examined under an optical microscope (Axioskop 40; Carl Zeiss, Göttingen, Germany) and photo-documented.