2.11. Immunohistochemistry

Immunohistochemistry for CD44, CD49f, P-cadherin, EpCAM, and ALDH1 was performed in 3-μm sections. Slides were placed in a Clear-Rite bath (Thermo Fisher Scientific, Waltham, MA, USA), rehydrated through a descending series of ethanol washes, and finally placed in distilled water. Epitope exposure was performed for 30 min at 95 °C with Tris/EDTA (Novocastra, Newcastle, UK) for CD49f and P-cadherin or citrate buffer (ThermoScientific, Freemont, CA, USA) for CD44, EpCAM, and ALDH1. Expression analyses were evaluated as follows: CD44 was detected using the antibody from Cell Signaling Technology (clone 156-3C11; Cell Signaling Technology, Danvers, MA, USA) (dilution 1:100); CD49f was assessed using the specific antibody from Sigma-Aldrich (HPA012696, Sigma-Aldrich, Darmstad, Germany) (dilution 1:50); P-cadherin expression was evaluated with the monoclonal antibody from BD Biosciences (clone 56, BD) (dilution 1:50); EpCAM was evaluated with the antibody from Santa Cruz Biotechnology (clone C-10, Santa Cruz Biotechnology, Dallas, TX, USA) (dilution 1:50); and ALDH1 was detected with an antibody from Abcam (clone EP1933Y, Abcam, Cambridge, UK) (dilution 1:100). Primary antibodies were detected using a secondary antibody with horseradish peroxidase polymer (Cytomation Envision System HRP; DAKO, Carpinteria, CA, USA) (CD49f, P-cadherin, EpCAM, ALDH1) or with the labeled biotin-streptavidin method (DAKO, Carpinteria, CA, USA) (CD44), according to the manufacturer’s instructions. The detection method used diaminobenzidine (DAB) as chromogen. Of note, the BCSC markers CD44, CD49f, P-cadherin, and ALDH1 were previously characterized in the primary breast cancer series [34,41,42]; thus, only the EpCAM marker was performed in this specific series, as described. For breast cancer brain metastases, all BCSC markers were evaluated in the context of this work.