IHC for COX-2, VEGF, and MMP-9 was performed according to the protocol described previously [40 (link)] from the lab. Briefly, the corneal sections were first rehydrated, then heat-mediated epitope retrieval was performed, endogenous peroxidase activity was blocked, and sections were incubated with the respective primary antibodies [anti-VEGF (Cat#ab28775), anti-MMP-9 (Cat#ab58803) both from Abcam, Cambridge, MA, and anti-COX-2 (cat#160112; Cayman chemicals, Ann Arbor, MI); Rabbit IgG antibody (N-Universal, DAKO), negative control]. Thereafter, the sections were incubated sequentially using the streptavidin-biotin complex (ABC) IHC staining methodology for signal amplification. DAB was used for visualization of IHC (brown, cytoplasmic staining) followed by hematoxylin staining for the nucleus. The scoring for the cytoplasmic staining was performed in 10 randomly selected areas (400x magnification). This intensity of IHC staining was scored from 0 to 4; where a score of 0 signified no DAB staining (absence of any brown color) and 4 signified the maximum intensity of staining, described previously [40 (link)].
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