Immpress peroxidase detection kit

Immunocytochemistry (ICC) was essentially as described (19 (link)). Primary antibodies as noted above CK5 (1:100), CK17 (1:200); vimentin (1:200), or CK8/18 (1:2000) for 2 h, secondary antibodies (A11029, A11037, A11008, A11032, Invitrogen, 1:200) for 1 h, and counterstained with DAPI. Cells were imaged using the Olympus BX40 fluorescent microscope and channels merged in Adobe Photoshop 2021. Immunohistochemistry (IHC) was performed as previously described (33 (link)) using CK5 (rabbit, ab75869, Abcam, 1:200), CK17 (rabbit, ab109725, Abcam, 1:100), vimentin (mouse, 5G3F10, Cell Signaling Technologies, 1:100), or pan-CK (mouse ab86734, 1:400, abcam, Waltham, MA) antibodies and either fluorescent secondary antibodies as described and imaged using an Olympus BX40 fluorescent microscope, or ImmPRESS Peroxidase detection kit (Vector Laboratories).

Immunohistochemistry and ICC were performed essentially as previously described.^{20 (link),56 (link)} Primary antibodies to CK5 (NCL-L-CK5, Leica Biosystems, 1:200), RARα (ab28767, Abcam, 1:100) and PR (PgR 1294, Dako, 1:500) were applied, followed by secondary antibodies, and developed using ImmPRESS Peroxidase detection kit (Vector Laboratories, Burlingame, CA, USA) for immunohistochemistry and secondary fluorescent antibodies (A11029, A11037, Invitrogen, Grand Island, NY, USA) for ICC. For immunohistochemistry, slides were scanned into the Aperio digital pathology system (Leica Biosystems) and whole sections analyzed for percent of positive cells using an algorithm tuned for CK5, therefore blinding was not necessary.