Triple negative breasts cancer (TNBC) is an aggressive breast cancer subtype for which there is a need to identify new therapeutic targets. of TNBC using the VECTRATM NKSF2 platform. There was a close relationship between nuclear and cytoplasmic ERβ1 expression. ERβ1 was expressed in a subset of TNBCs but its expression was significantly associated with Ki67 in only one of the cohorts. There was no significant association between ERβ1 expression and disease-free and overall survival in either cohort. Although these results suggest that ERβ1 expression alone may not be informative in TNBCs this study provides a new strategy for optimizing and objectively measuring ERβ1 expression in tissues which may provide a standard for ERβ1 immunohistochemistry in future large-scale clinical studies aimed at better understanding the role of ERβ1 in breast cancer. studies have shown that the two ERs regulate proliferation in opposite manners. While ERα stimulates proliferation in response to estrogens ERβ expression and activation by estrogens has been shown to inhibit SVT-40776 the growth of both ERα-positive and ERα-negative breast cancers [6-10]. Because of the consistent data demonstrating the antiproliferative activity of ERβ it has been suggested that ERβ may act as a tumor suppressor and could be a possible therapeutic target for cancers such as TNBC. ERα and ERβ are expressed from unique genes on separate chromosomes. The gene can encode several different ERβ isoforms. The full-length isoform ERβ1 is the only isoform that has a high affinity for 17β-estradiol (E2) and can transactivate gene expression in response to ER ligands [11 12 Four additional ERβ isoforms ERβ2-ERβ5 have been identified in human tissues . These isoforms have unique C-terminal sequences that arise from alternative splicing through the seventh exon from the gene. Although these isoforms don’t have high affinity for ER ligands ERβ2-ERβ5 possess the capability to dimerize with ERβ1 to improve transactivation in response to ER ligands . Because ERβ1 may be the just isoform with the capability to bind ligands with high affinity this receptor will be the principal isoform to mediate gene manifestation and development inhibition in response to E2 or ERβ-selective ligands. Certainly just the full size ERβ1 isoform inhibited SVT-40776 the development of ERα-adverse breasts cancers SVT-40776 cells [3 8 In light of the consistent evidence suggesting that ERβ1 is usually antiproliferative several studies have aimed to assess the clinical significance of ERβ1 expression in breast cancers; however the data have been inconclusive [14-21]. The receptor should ideally be detected at the protein level as a poor correlation between ERβ1 mRNA and protein has been observed in SVT-40776 breast cancers . However the antibodies and cutoffs used to determine ERβ1 expression have been inconsistent across studies  and there is a need to stringently confirm the specificity of ERβ1 immunohistochemical protocols. In this report we describe a system to optimize immunohistochemistry (IHC) for full length ERβ using xenograft tissue obtained from breast cancer cell lines with inducible expression of ERβ1 . We used these protocols to assess the subcellular localization of ERβ1 in two cohorts of patients with Stage IIII TNBC. We also decided the associations between ERβ1 and the proliferative marker Ki67 as well as tumor grade tumor stage and survival. This study provides a new strategy for optimizing ERβ1 IHC and objectively detecting the nuclear localization of the receptor which may prove useful for future clinical studies aimed at determining the importance of full length ERβ expression in breast cancers. Materials and methods Cell lines and reagents HEK293 cells were cultured in cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen Carlsbad CA). Turbofect and SuperSignal West Pico enhanced chemiluminescent reagent were obtained from Thermo Scientific (Rockford IL). Blasticidin S and Zeocin were purchased from Research Products International (Mount Prospect IL) and doxycycline was purchased from Clontech (Mountain View CA). Athymic nude mice were purchased from Harlan Laboratories (Madison WI). All the reagents for immunohistochemistry were purchased from Biocare Medical (Concord CA). A rabbit polyclonal antibody for ERα (HC-20) and a rabbit polyclonal antibody raised against the N terminus of ERβ (H150) were purchased from Santa Cruz Biotechnology (Santa Cruz CA). A polyclonal antibody raised against a peptide corresponding to the C terminus of.