Histopathological Evaluation of Pigmented Tumor Tissues

For histopathological evaluation, H&E staining was performed on paraffin-embedded tumor tissue sections. The degree of pigmentation of tissue samples was assessed based on the Collaborative Ocular Melanoma Study (COMS) classification system [49 (link)]. For immunohistochemistry, sections were stained with primary antibodies overnight at 4 °C. On the next day; sections were incubated for 30 min in ImmPRESS AP Reagent (Vector Laboratories, Burlingame, CA, USA), followed by incubating for 2–15 min in ImmPACT NOVA-RED (Vector Laboratories). The following primary antibodies were purchased from Agilent (Santa Clara, CA, USA): Melanosome (Clone HMB45), Melan-A, S100, and Ki67. All sources of specific antibodies for these markers and their dilution rates have been previously described [42 (link)] except for Ki67 (dilution rate; 1:50). Staining data for Ki67 were quantitated by counting all positive nuclei per field of vision at ×400 magnification using ImageJ software (available at http://rsb.info.nih.gov/ij) [40 (link)].