Anti erα

Cultured cells were grown on glass coverslips and fixated in ethanol 96%. Antigen retrieval was done by autoclaving in EDTA (0.1 M, pH 9.0). Slides were blocked with immunodetector peroxidase blocker (Bio SB, Santa Bárbara CA, USA). For EAG1, additional blocking was performed using background Sniper (Biocare Medical, CA, USA). The following primary antibodies were incubated for 2 hours: Anti- ERα (1:250, Bio SB), anti-VDR (1:100, Santa Cruz Biotechnology Inc, CA, USA) and anti-EAG1 (1:300, Novus Biologicals CO, USA). After washing, the slides were sequentially incubated with immuno-Detector Biotin-Link and immuno-Detector HRP label (Bio SB) during 10 minutes each. Staining was completed with diaminobenzidine (DAB) and slides were counterstained with hematoxylin.

Cultured cells were grown on glass coverslips and fixated in 96 % ethanol. Antigen retrieval was done by autoclaving in Retriever EDTA (Bio SB, Santa Bárbara CA, USA). Slides were blocked with immunodetector peroxidase blocker (Bio SB). The following primary antibodies were incubated for 1 h: Anti- ERα (1:250, Bio SB), anti-VDR (1:100, Santa Cruz Biotechnology Inc, CA, USA), anti-HER2 (1:100, Cell Signaling Technology, Beverly, MA) and anti-epidermal growth factor receptor (EGFR, 1:100, Bio SB). After washing, the slides were sequentially incubated with immuno-Detector Biotin-Link and immuno-Detector HRP label (Bio SB) during 10 min each. Staining was completed with diaminobenzidine (DAB) and slides were counterstained with hematoxylin.