Immunocytochemistry for Breast Cancer Markers

For immunocytochemistry (ICC), 1–2 × 10⁵ cells were seeded onto glass coverslips in 6-well plates in regular media. For assessing PR, cells were treated with 10 nM E2 for 48 h prior to collection. Cells were washed twice with PBS and fixed with ice-cold 70% acetone/30% methanol for 5 min. Fixed cells were blocked with 10% normal goat serum (Vector Labs, Burlingame, CA) in 0.05% TBS-T for 30 min. Primary antibodies were as follows: ERα (SP1, 1:200, Thermo-Fisher, Waltham, MA), PR (1294, 1:50, [26 (link)], CK5 (ab75869, 1:400, Abcam, Cambridge, MA), CK8/18 (NCL-L-5D3, 1:2000, Leica Biosystems), and Vimentin (5741, 1100, Cell Signaling, Danvers, MA) for 1 h. Secondary antibodies were A11029 (green) and/or A11037 (red) (Invitrogen, 1:200) for 30 min followed by counterstaining with 0.1 μg/mL DAPI. Phase contrast images were captured using a Nikon TiE microscope (Nikon, Melville, NY) equipped with a digital camera and NIS Elements 4.6 software. Fluorescent images were captured using an Olympus BX40 microscope equipped with a digital camera and cellSens Standard 1.13 software. Adobe Photoshop CC 2019 was used to perform minimal linear adjustments to brightness/contrast and to assemble pictures into multipanel figures. Immunohistochemistry (IHC) of PDX was performed with the same antibodies for the indicated markers as previously described [21 (link)].