Immunohistochemistry Protocol for Breast Cancer Markers

IHC was performed as previously described.^{28 (link)} Briefly, 10 μm paraffin sections were subjected to antigen retrieval in citrate buffer. Primary antibodies used were as follows: ERα (SP1, ThermoFisher, 1:100), PR (1294, DAKO, 1:500), CK5 (NCL-L-CK5, Leica Biosystems, 1:500), CK8/18 (NCL-L-5D3, Leica Biosystems, 1:500), and CA2 (SAB2700301, Sigma, 1:1600). Secondary antibodies were anti-Rabbit Ig MP-7401 (Vector, 1:2000; Burlingame, CA, USA) or anti-Mouse Ig MP-7402 (Vector, 1:2000). Development used the ImPACT DAB Peroxidase Substrate Kit (Vector). Images were captured using the Aperio Digital Pathology system (Leica Biosystems) and assembled in Adobe Photoshop CC with minimal linear changes to contrast. Quantification of the percentage of positive cells was performed using the Imagescope software (Leica) that used algorithms tuned for nuclear or cytoplasmic staining.