Pa5 28647

Esophageal carcinoma tissue microarrays were purchased from Bioaitech Co., Ltd. (Xi’an, China; ethical license: 2005DKA21300), which contained a total of 148 specimens consisting of EAC (n = 50), ESCC (n = 78), and normal esophagus (NE) tissues (n = 20). The streptavidin–peroxidase method was used for immunohistochemistry analysis of the tissue microarrays. The slides were stained with the GPER1 antibody (PA5-28647; Invitrogen, Carlsbad, CA, USA) [12 (link),52 (link),53 (link)] and visualized with DAB after incubation with the secondary antibody.
The staining results were analyzed using a Vectra Multispectral Imaging System (Perkin Elmer, Waltham, MA, USA). Vectra 2.0.8 (Perkin Elmer) was used for scanning the slides. After importing the high-magnification images and spectral information in inForm 1.2 (Perkin Elmer), positive cells were identified in the target area and the positive rate was determined by setting the threshold intensity. The expression of GPER1 in the tissue microarray is shown with an automatically calculated H-score (=Σpi*i, where i means staining intensity, i = 0,1,2,3; pi is the percentage of the number of cells with corresponding staining intensity in the overall cells). Positive staining was defined as an H-score ≥ 100 (maximum = 300) in this study.

The tissue sections (7 μm thick), after deparaffination and rehydration, were incubated in 3% H₂O₂ for 20 min at room temperature (RT). After blocking the non-specific sites in 1.5% normal goat serum (NGS) diluted with phosphate-buffered saline (PBS) pH 7.2 (NGS cod S1000, Vector Laboratories, Burlingame, CA, USA), sections were incubated with rabbit polyclonal anti-GPR30 antibody (dil. 1:500, cod. PA5-28647, Invitrogen, Waltham, MA, USA), mouse monoclonal anti-ESR1 (dil. 1:200, cod. TA807239, OriGene Technologies Inc., Rockville, MD, USA), and rabbit polyclonal anti-ER-beta (dil. 1:500, cod. PA1-311, Invitrogen, Waltham, MA, USA) overnight at 4 °C.
After washing in PBS, the sections were incubated with HRP-conjugated secondary antibody (dil. 1:4, cod. UNIHRP-050 ImmunoReagents, Raleigh, NC, USA) for 30 min at RT. The immunoreactions were developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) (SK-4100, Vector Laboratories, Burlingame, CA, USA). In addition, the sections were counterstained by using hematoxylin. The negative controls included omission of primary antibody (Supplementary Materials).
The immunoreactions were observed by two different blind observers who then captured images and analyzed them using a Leica DM 6B light microscope and SFC7000T digital camera.