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 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.
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