Keratin 14

Only cells without fluorescent protein tags were stained. Cells were fixed with ice-cold methanol/acetone (50/50 v/v) for 10 minutes at room temperature. The cells were incubated at 4°C overnight with the following primary antibodies: keratin 14 (RB-9020, 1:4,000), keratin 18 (MS-142, 1:2,000), keratin 19 (MS-198 1:1,000), β-casein (MS-935 1:1,000), Ki67 (RB-1510, 1:500) (Thermo Scientific, Stehelin), p63 (MS-1081, 1:200) (Thermo Scientific), e-cadherin (610181, 36/Ecad, 1:1,000) (BD Bioscience,), EpCAM (Clone 9C4, 1:100) (BioLegend), or keratin 8/18 (GP11, 1:500) (Fitzgerald). Goat anti-mouse, goat anti-rabbit or goat anti-guinea pig secondary antibodies coupled to Alexa 488, 568 or 633 (Molecular Probes, Invitrogen 1:500) were used for detection.

Cytospin preparations (20,000 cells per slide) were prepared as previously described [14 (link), 20 (link)], fixed with 10% neutral-buffered formalin (NBF) for 10 min at room temperature, and stained for Keratin 5 (Covance Innovative Antibodies, Rabbit, 1:1000) and Keratin 14 (Thermo Scientific, Mouse, 1:500). Slides were counterstained with 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI). Images of at least 200 nucleated cells were acquired using Zeiss Imager.Z1 fluorescent microscope (Zeiss) and quantified using ImageJ software.

Tissues were dissected, fixed in 4% paraformaldehyde and incubated overnight at 4 °C with primary antibodies. The following day, tissues were incubated with secondary antibodies. Tissues were subsequently incubated in 80% glycerol before dissection for three-dimensional imaging^{64 (link)}. The following primary antibodies were used: Keratin 14 (rabbit polyclonal, Thermofisher, cat#: LBVRB-9020-P0, 1:500 dilution), Keratin 8/K18 (rat, clone: TROMA-I, DSHB, 1:500 dilution), and Vimentin (mouse, clone LN-6, Sigma cat# V2258, 1:200 dilution). All secondary antibodies were Alexa Fluor-conjugated: anti-rabbit Alexa Fluor 488 (Invitrogen; 1:500 dilution), anti-mouse Alexa Fluor 555 (Invitrogen; 1:500 dilution), anti-rat Alexa Fluor 647 (Invitrogen; 1:500 dilution).