Publication protocol
"The samples were cut into 4µm thick serial sections, one of which was stained with
hematoxylin and eosin, and the others were used for immunohistochemistry.
Immunohistochemistry was carried out for a panel of markers, including CK7, CDX2, HER2, and HER4,using a Leica Bond Max automated slide staining system
(Leica, Bannockburn,IL). The sections were counter-stained with hematoxylin.
For CK7, staining of the cytoplasm, the cell membrane, or both was considered positive. CDX2 expression with nuclear and strong staining was regarded as positive. Membrane staining was evaluated for HER2. Staining of HER2 was classified into four categories (0, no reactivity or membranous reactivity in less than 10% of tumor cells; 1+, faint or barely visible membranous reactivity in more than 10% of cells; 2+, weak to moderate
complete or basolateral membranous reactivity in more than 10% of cells; and 3+, moderate to strong complete or basolateral membranous reactivity in more than 10%
of cells) according to the immunohistochemistry (IHC) scoring system for gastric tumors created by Hofmann et al. (15). Cytoplasmic staining was also evaluated for
HER4. Positive staining for HER4 was considered when at least 10% or more of tumor cells were stained, and cytoplasmic staining intensity was scored and graded
from 0 to 3 (16). We considered only IHC 3+ staining as overexpression of HER2 and HER4 (Figure 2)."
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