E p1932y

Tumor samples were distributed in 15 TMAs using 4 mm tissue cores at the Experimental Pathology Laboratory of Pontifical Catholic University of Paraná (PUCPR). From each donor block was extracted one or two cylinders 4 mm in diameter and deposited in the receiver blocks, previously prepared. In each TMA, a sample of normal breast tissue was included as an internal control. Thereafter, 4 μm tissue sections from the TMA blocks were transferred to electrically charged Star Frost® (Braunschweig, Germany) slides and incubated with primary antibodies [anti-CD44 (anti-human mouse monoclonal, clone DF1485, dilution 1/40, Novocastra, Newcastle, UK), anti-CD44v6 (antihuman mouse monoclonal, clone VFF-7, dilution 1/100, Novocastra, Newcastle, UK), anti-CD24 (anti-human rabbit polyclonal, dilution 1/200, Abbiotec, San Diego, CA, USA), and anti-ALDH1 (anti-human rabbit monoclonal, clone E-P1932y, dilution 1/100, Epitomics, Cambridge, Massachusetts, USA)] for 12 h in a humidified chamber at 2−8°C. An Advance Dako (Caripenteria, CA, USA) secondary antibody was incubated with the slides for 30 min at 2−8°C. The reactions were developed using a DAB chromogen-substrate solution (Dako). Harris hematoxylin was used for counterstaining. Positive and negative (incubated without primary antibody) controls were run in parallel with all reactions.