Hpa029424

Multiplex IHC staining was performed to examine the co-expression of ATAD2 and Ki-67 in OC tissue sections on a commercially obtained OC tissue microarray (OV241c; containing 12 primary OC tissues) from Alenabio using an Opal™ 4-Color Manual IHC kit (PerkinElmer, Inc.). Briefly, the sections were first incubated with a specific ATAD2 antibody (1:1,000; catalog no. HPA029424; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, followed by incubation with a goat anti-rabbit immunoglobulin G antibody (1:5,000; catalog no. NEL840001KT, PerkinElmer, Inc.) for 1 h at room temperature and subsequently labeled with Opal 520 fluorophore (PerkinElmer, Inc.). For Ki-67 staining, an anti-Ki67 antibody (1:1,000; catalog no. ZM-0166; OriGene Technologies, Inc.) was used at room temperature for 1 h and Opal 690 fluorophore (PerkinElmer, Inc.) was used for labeling. Finally, nuclei were stained with DAPI (PerkinElmer, Inc.) for 30 min at room temperature and mounted with anti-fade medium. The inForm 2.4.2 software (PerkinElmer, Inc.) was used to analyze the multispectral images.

A standard IHC protocol was performed on a commercially obtained OC tissue microarray (OV802a) containing 60 primary OC tissues, 10 tissues of stomach metastases to the ovary and 10 adjacent normal tissues from Alenabio to assess the protein expression levels of ATAD2 in OC. Following IHC staining with a specific antibody against ATAD2 (1:500; catalog no. HPA029424; Sigma-Aldrich; Merck KGaA) overnight at 4°C, the samples were stained with a horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (1:2,500; catalog no. ZB-2301; OriGene Technologies, Inc.) for 1 h at room temperature. The immunohistochemical signals were then observed under a light microscope (magnification, ×200). The staining intensity was scored as follows: 0, Negative; 1, weak; 2, moderate; and 3, strong. The percentage of ATAD2-positive cells was also scored as follows: 0, 0-10%; 1, >10-25%; 2, >25-50%; 3, >50-75%; and 4, >75-100%. The final immunoreactive score was determined by multiplying the intensity and percentage scores, with a minimum score of 0 and a maximum score of 12.