Ck8 18

The formalin-fixed, paraffin-embedded (FFPE) tissue block specimens were sectioned (3-μm thick), deparaffinized and rehydrated. Each section was stained with hematoxylin and eosin and then used for immunostaining. Immunohistochemical analyses were performed using an autostainer (BenchMark XT system; Ventana Medical Systems, Inc., Tucson, AZ, USA) according to the manufacturer’s instructions. The following anti-human primary antibodies were used: p63 (Santa Cruz Biotechnology, Inc,. Santa Cruz, CA, USA), caldesmon, calponin, α-smooth muscle actin, CD10, cytokeratin (CK) 5/6 (DakoCytomation, Glostrup, Denmark) and CK8/18 (Novocastra, Newcastle, UK) (Table I).
Stained sections were evaluated by two different pathologists using uniform criteria. Discrepancies were resolved through simultaneous evaluation and discussion of the results. Single-marker expression was recorded as negative/positive and high/low level, following consideration of the expression in reactive surrounding tissue compared with tumoral cells and the specific cut-off of each marker.

Immunohistochemistry was performed on tumors as previously described¹³ . Unmasking was performed using citrate buffer, pH 6.0. Antibodies used were Cx43 (Abcam, ab11370), ERα (SP1, Thermofisher, RM-9101), CK8/18 (Novocastra, NCL-5D3), Ki-67 (Novus Biologicals, NB500-170), and c-KIT (R&D Systems, AF1356). Images were captured using an Olympus BX40 microscope equipped with an Olympus DX73 camera and Cell Sens software. For Ki67 staining images were captured using the Aperio Digital Pathology system (Leica Biosystems), and samples were quantified using Imagescope software (Leica Biosystems). Mucicarmine and H&E staining were performed as previously described¹³ .
LASER Capture Microdissection, microarrays on tumors: Five million BCK4 cells were injected into the 4th mammary gland of NOD-SCID mice under an approved IACUC protocol (83913(12)1E) and grown for 5 months. Tumors were resected, embedded in O.C.T (Tissue-Tek), frozen in N2, and sectioned on a cryostat. Two tumors were used to obtain duplicate samples. Three thousand cells from each tumor region were LASER captured using the ArcturusXT microdissection system, RNA was isolated using the PicoPure RNA isolation Kit (Arcturus) followed by cDNA preparation using the Ovation Pico WTA System and Encore Biotin Module kit as previously reported^{59 (link)}. Affymetrix Human Genome U133 Plus 2.0 Arrays were used to measure gene expression.