Ti ds

The expression of FUT4 was analyzed by immunohistochemistry (IHC) using paraffin-embedded melanoma patient tissues. Serial sections (4 μm each) were prepared, deparaffinized in xylene and rehydrated in a graded alcohol. After microwaved for 20 min in citrate buffer to expose the antigen and washed with PBS, slides were incubated in 3% H₂O₂ for 10 min at room temperature to block endogenous peroxidase activity. Non-specific binding was blocked with goat serum at room temperature for 30 min before incubation overnight at 4°C with rabbit IgG FUT4 (1:100). After extensive washing with PBS, sections were incubated with respect secondary antibody for 30 min at room temperature. The signal was visualized with peroxidase-labeled streptavidin complexes DAB and the sections were briefly counterstained with hematoxylin. Yellowish-brown stain indicated a positive result. The negative control was generated by replacing the primary antibody with isotype IgG. Slides were mounted and visualized on an inverted microscope (Nikon Ti-DS, Japan).

Paraffin-embedded gastric tissues were analyzed by immunohistochemistry (IHC). Serial sections (4–6 mm each) were deparaffinized in xylene and rehydrated in a capsaicin concentration gradient and evaluated with antibodies to detect H. pylori (1:100), pepsinogen I and II (1:100 each), MPO (1:100), F4/80 (1:100), CD-34 (1:100), PCK-26 (1:100), and SMA-α (1:100). Sections were subsequently incubated with their respective secondary antibodies for 30 min at room temperature. The signal was visualized with peroxidase-labeled streptavidin complexes by DAB, and the sections were briefly counterstained with hematoxylin. The immunohistochemical localization pattern was also recorded by digital imaging (Nikon Ti-DS, Tokyo Japan). The ImageScope (11.1.1.752) software program was used, and the labeling index was calculated as a percentage of positive cells relative to the total number of counted cells.

Commercially available patient tissue arrays from US Biomax Inc. (Rockville, MD), were used for IHC. For mouse studies, paraffin-embedded melanoma tissues were analyzed by immunohistochemistry (IHC). Serial sections (4–6 mm each) were deparaffinized in xylene and rehydrated in an alcohol concentration gradient and evaluated with antibodies to detect TIM (1:200), TIPIN (1:30 each), and PCNA (1:200). Sections were subsequently incubated with their respective secondary antibodies for 30 min at room temperature. The signal was visualized with peroxidase-labeled streptavidin complexes and DAB, and the sections were briefly counterstained with hematoxylin. The immunohistochemical localization pattern was also recorded by digital imaging (Nikon Ti-DS, Japan). The IOD value of each tumor tissue was analyzed from 3 different fields using the Image-Pro PLUS (v.6) computer software program following an established protocol.