Ab288

Human breast cancer tissue specimens (HBreD070CS02) were purchased from Shanghai Outdo Biotech CO. The tissues (n = 70) including 11 paracancerous tissues, 4 intraductal carcinoma and 55 invasive ductal carcinoma. The primary antibodies against ERβ (ab288, 1:200, Abcam), CLDN6 (V118, 1:200, Bioworld Technology) and beclin1 (D40C5, 1:200, CST), respectively, and then incubated at 4 °C overnight in a humidified container. Following washing three times with phosphate buffer saline (PBS), the section was treated with UltraSensitive™ SP (Mouse/Rabbit) IHC Kit according to the manufacturer’s instructions (KIT-9710, MXB Biotechnology, Fuzhou, China). 3, 3′-diaminobenzidine (DAB) was used for color development. Immunostaining was evaluated by two pathologists using a blind protocol design. For ERβ, CLDN6 and beclin1, each tissue sample was scored according to its staining intensity (0, none; 1, weak; 2, moderate; 3, strong) multiplied by the point of the percentage of stained cells (positive cells ≤25% of the cells: 1; 26–50% of the cells: 2; 51–75% of the cells: 3; ≥75% of the cells: 4). The range of this calculation was 0–12. The median value of scores was employed to determine the cut-off. Cancers with scores above the cut-off value were considered to have high expression of the indicated molecule and vice versa.

The expression of estrogen receptor (ER)-α,ER-β, and progesterone receptor (PR) in gastric cancer, which may potentially be related to the ovarian metastasis, was detected by immunohistochemistry (IHC) using the Novolink Polymer Detection System as described previously [10 (link)]. The following primary antibodies were used: ER-α (ab37438, dilution 1:200; Abcam, Cambridge, UK), ER-β (ab288, dilution 1:100; Abcam), and PR (ab16661, dilution 1:100; Abcam). All specimens were independently evaluated by two pathologists who were blinded to study grouping. Samples where more than 10% of the tumor cells were stained were regarded as positive.

Cells were fixed with 4% formaldehyde (Sigma) in PBS for 10 min followed by permeabilization with 0.2% (w/v) Triton in 1× PBS for 15 min. Non-specific protein binding was blocked by incubation with 5% goat serum in 1× PBS-T (PBS + 0.02% w/v Tween 20) for 20 min at room temperature. Cells were incubated with anti-cytokeratin 19 (CK19) (Abcam, ab 84632) plus anti-ERα (Abcam, ab2746) or anti-ERβ (Abcam, ab288) primary antibodies in blocking buffer at 4 °C overnight. Following three 10-min washes in PBS-T, cells were incubated with the appropriate secondary antibodies (Cell signaling, 4412S and 4414S) in blocking buffer for 45 min in the dark. Cell nuclei were stained with DAPI prior to mounting using Fluoroshield mountant (Sigma).