NAPSIN A (TMU-AD02) ANTI-HUMAN MOUSE IGG MOAB

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Immunohistochemistry was performed at the immunohistochemistry laboratory at the University of Texas Health Science Center (San Antonio, Texas). Antibodies used included anti-human Nap-A mouse immunoglobulin G (IgG) monoclonal antibody (TMU-Ad02, 1∶100; Immuno-Biological Laboratories Co, Ltd, Takasaki, Japan) and anti-rat TTF-1 mouse IgG monoclonal antibody, known to react with human TTF-1 (8G7G3/1,1 1∶80; DakoCytomation Inc, Carpinteria, California). Protocols, reagents for antigen retrieval (Reveal Decloaker 10×), and the detection system were from Biocare Medical (Concord, California). Slides were stained with a Ventana Immunostainer (Ventana Medical Systems, Tucson, Arizona). Both Nap-A and TTF-1 immunostaining were performed on the first group of tissue specimens (from Toyama University Hospital). Only Nap-A immunostaining was performed on the second group of tissue specimens (from Weill College of Medicine). After staining, the tissue microarrays were scanned with a Spectrum/Spectrum Plus (Aperio Technologies, Inc; Bristol, United Kingdom) to a dedicated server and were analyzed by 3 pathologists independently. Staining intensity was evaluated as follows: negative, no staining to minimal light-brown to dust; weak positive, minimal, patchy, or diffuse cytoplasmic staining for Nap-A or nuclear staining for TTF-1; and strong positive, moderate to intense-brown, granular, cytoplasmic staining for Nap-A or nuclear staining for TTF-1. All results were evaluated relative to a negative control of the same tumor.

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