Decreased expression of eukaryotic initiation factor 3f is an adverse prognostic factor for stage I–III gastric cancer

Li, Guanghua; Wang, Na; Sun, Chuanjin; Li, Bo

The polyclonal antibody (ab64177) to eIF3f was bought from Abcom (Cambridge, UK). The paraffin-embedded tissue blocks were sectioned in 3 to 4 mm slices and placed on Anti slides. After de-waxing and hydration, the slides were rinsed in phosphate-buffered saline (PBS) and blocked for 10 min with 3% hydrogen peroxide to deprive the endogenous peroxidase activity. After antigen retrieval with the use of a microwave, the specimens were incubated with the anti eIF3f MAb (diluted 1:100 in PBS) at 37°C for 1.5 hours. The sections were then washed 3 × 3 min in PBS and incubated with 1 and 2 Reagent of PV9000 Mouse/Rabbit hypersensitivity two-step immunohistochemical Kit (Beijing fir Jinqiao, Beijing, China) for a total of 60 min at 37°C in a humid chamber. The sections were washed 3 × 3 min with PBS, followed by the addition of diaminobenzidine as a chromogen for 3 to 5 min, which was strictly controlled under a microscope. Antibodies were optimized using a positive control tissue according to the manufacturer's instructions. In negative controls, the primary antibody was replaced with PBS. The remaining procedures were performed in parallel with other specimens. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria at × 100 and × 200 magnification. The overall percentage of positive cells on an immunostained section was determined according to the pattern of intracellular localization. The extent and pattern of the eIF3f-specific immunostaining within a tissue section were determined by the percentage of cells with cytoplasm staining. The immunostaining was read in a semiquantitative manner. Three visual fields were examined randomly and the rate of positive cells was divided into less than 5% (score 0), 6% to 25% (score 1), 26% to 50% (score 2), 51% to 75% (score 3), and more than 75% (score 4). The staining intensity can be divided into three grades: no staining (score 0), slightly yellowish (score 1), brownish yellow (score 2), and dark brown (score 3). The multiplication of the two were graded as follows: 0 (score 0), 1+ (score 1-4), 2+ (score 5-8), and 3+ (score 9-12). Intensity scores of 0 or 1+ were designated as low expression and 2+ or 3+ were designated as high expression.