Krt19

Array-bound and well-bound cells were fixed in 2% PFA for 15 minutes at RT following respective treatments. Cells were then permeabilized with .3% Triton X-100 for 25 minutes at RT. Array-bound cell primary antibody staining was performed with KRT14 (Abcam, 1:200), KRT19 (Dako, 1:200), and DAPI (ThermoFisher, 1:10,000). Secondary antibody staining was performed with IgG3 Alexa Fluor 488 (ThermoFisher, 1:200), and IgG1 Alexa Fluor 555 (ThermoFisher, 1:200). Only DAPI and EdU detection was performed on well-bound cells, with the exception of Figure 2B. Well plates were imaged on the GE InCell 6000 platform, and image analysis and cell count quantification were performed on the GE InCell Analyzer software package. Size gating of nuclei was used to exclude apoptotic cells, and EdU positivity was determined as nuclei having a mean fluorescent intensity above an experimentally consistent threshold (this threshold was defined using single cell parametric analysis plotting total DAPI intensity against mean EdU intensity). All fluorescent imaging studies were performed at consistent intensity and gain settings across experiments.

Cells were fixed with formalin and centrifuged at high speed in fluid agar. Agar-containing cell pellets were subsequently embedded in paraffin blocks which were cut into 4-μm sections. Primary antibodies against keratin 19 (KRT19, 1:100; Dakocytomation, 1:250; Abcam) and GFP (1:500; Roche and Molecular probes) were used. Secondary antibodies were Alexa-Fluor 488 and Alexa-Fluor 568 anti-mouse or anti-rabbit (1:1000; Molecular Probes), 4′,6-diamidino-2-phenylindole (DAPI) (Vector) was used for nuclear counterstaining. Immunofluorescent images of paraffin slides were acquired with a DM5500 microscope (Leica). The GFP expression of live cells was evaluated with a CK40 microscope (Olympus) using endogenous GFP expression. Images were processed using Zen Lite software (Zeiss).