Aperio digital pathology system

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.

All the lung lobes were dissected and processed for paraffin embedding. Five micron paraffin sections were stained with hematoxylin and eosin and slides were scanned with the Aperio™digital pathology system (Aperio; Leica Biosystems, Wetzlar, Germany). The lung area per section was measured using Aperio Scanscope (Leica Biosystems) and the metastatic burden (mm² of metastases/cm² lung) was calculated for each animal, using the data from three sections at least 100 μm apart, as previously described [39 (link)].

Tissues were fixed in 10% neutral-buffered formalin, and processed and embedded according to standard histologic protocols. Five-micron sections were stained with hematoxylin and eosin (H&E) or used for immunohistochemical analysis. The GLUT1 antibody has been described previously [31 (link)]. Anti-Cre recombinase antibody was from Thermo Scientific, anti-Ki67 antibody was obtained from Dako, and anti-BrdU antibody was from Abcam. Cre recombinase and Ki67 were quantified by counting the number of positive cells out of the total number of mammary epithelial cells in one full, representative mammary gland section per mouse. BrdU staining was quantified by counting the number of positive cells out of the total cells per acinar structure in 3–4 paraffin-embedded sections per treatment, cut every 50 μm. GLUT1 and tumor area were quantified using the Aperio Digital Pathology system (Leica Biosystems).

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.