Nbp1 52193

To evaluate mucin mRNA expression at the protein level, tissue segments were fixed for 24 h in 4% formaldehyde and subsequently embedded in paraffin. Five-micrometer cross-sections were deparaffinized, rehydrated, and used for immunohistochemical staining using target-specific primary antibodies and visualization with a secondary streptavidin–horseradish peroxidase antibody and 3-amino-9-ethylcarbazole (AEC) substrate to detect the expression and localization of MUC1 (AF6298, R&D systems, 1:500), MUC2 (NBP1-31231, Novus Biologicals, 1:3000), MUC4 (NBP1-52193, Novus Biologicals, 1:3000), MUC5AC (ab3649, Abcam, 1:5000), MUC6 (ab216017, Abcam, 1:50), and MUC13 (MABC209, Merck Millipore, 1:1000). The stained sections were analyzed by light microscopy (Olympus BX43) [30 (link)]. Distinct staining in more than 10% of the gastric cells was recorded as positive immunoreactivity for the relevant mucin. Tumors containing epithelial cells expressing only gastric or intestinal-type mucins were classified as having a gastric or intestinal mucin phenotype, respectively. Those containing cells expressing both gastric and intestinal type mucins were classified as having a mixed phenotype, whereas tumors with cells expressing neither gastric nor intestinal type mucins were classified as having a null mucin phenotype. The degree of immunostaining was evaluated by two independent observers.