Biotinylated rabbit anti mouse antibody

To detect YB-1 protein expression in human breast cancer and adjacent tissues by immunohistochemical staining, 5-μm-thick sections were placed on precoated slides with 3-(triethoxysilyl)propylamine (Merck, Darmstadt, Germany). The Slides were soaked in xylol for 1 h and washed in a series of alcohols with decreasing concentrations. After tissue deparaffinization, antigen retrieval was performed on the sections in a microwave for 5 min in TEC buffer (0.05 M Tris-HCl, 0.05 M ethylenediaminetetraacetic acid, 0.02 M Na-citrate (pH 7.8)), followed by blocking in peroxidase. After incubation with a primary antibody against YB-1 for 12 h, the slides were incubated with a biotinylated rabbit anti-mouse antibody (Vector, Grünberg, Germany) for 30 min. Subsequently, the slides were stained with diaminobenzidine (DAB) (Sigma, USA) for 10 min to label YB-1 proteins and then counterstained with hematoxylin to label nuclei. Cancerous and paracancerous tissue from patients with ER-positive breast cancer were obtained from Shenzhen Second People's Hospital, Shenzhen University. All human breast cancer sample acquisitions were approved by the Committee on Ethics of Shenzhen Second People's Hospital, Shenzhen University. Written informed consent was obtained from all the participants.