Axiocam 305 color camera

Coverslips with PFA-fixed cultures were washed in PBS three times for 15m each. Next, cultures were permeabilized with 0.1% Triton X for 10m at room temperature and washed in PBS three times for 15m each. After blocking for 1h at 37° C in 10% donkey serum in PBS, primary antibodies (Table 1) were applied and incubated at 4° C overnight. The next morning, cultures were washed three times in PBS for 15m each and secondary antibodies were applied. After 30m incubation at 37° C, slides were washed three times in PBS for 15m each. After removing excess PBS, coverslips were mounted to slides with Fluorogold II with DAPI (Electron Microscopy Sciences, Hatfield, PA). Slides were imaged on a Zeiss Axios Scope A1 Microscope (Zeiss, Jena, Germany) with a Zeiss Axiocam 305 color camera (Zeiss) in the University of Georgia’s microscopy core at 10 or 20x magnification. ImageJ (US National Institutes of Health, Bethesda, MD) cell counter plug-in was used to mark cells as they were counted and the proportions of cells positive for each stain or combination were determined from total DAPI counts. Antibodies selected are well-referenced in the literature (except for SOX10; validation images are provided on the manufacturer’s website) and antibody specificity was confirmed by 1) double staining with other cell specific markers, and 2) omitting the primary antibody resulting in lack of staining.

Cell morphology was assessed using brightfield microscopy on living cells at various densities. For IF microscopy, cells were washed with PBS and then fixed with 10% neutral buffer formalin (NBF) (Thermo Fisher Scientific) for 15 min at room temperature (RT). Cells were then incubated in Serum-Free Protein Block buffer (Agilent, Santa Clara, CA) supplemented with 0.1% Saponin (Sigma-Aldrich) for 30 min and subsequently in various primary antibodies diluted in Antibody Diluent with Background Reducing Components (Agilent) and 0.1% Saponin for 60 min, both at RT. Cultures were then washed thrice in PBS and incubated in secondary antibody for 60 min. After three washes in PBS, nuclei were counterstained with DAPI (1:1000) (Thermo Fisher Scientific) for 15 min. The DAPI stain was removed by three PBS washes. Primary antibodies used were: TE-7 (1:200, EMD Millipore, CBL271, Burlington, MA), vimentin (1:200, Abcam, Ab8978, Cambridge, MA) and ACTA2 (1:200, BioLegend, 904601, San Diego, CA). One secondary antibody was used as well (Alexa Fluor 546, 1:1000, Thermo Fisher Scientific, A11030). All cell images were collected using a Zeiss LSM 900 confocal microscope with a Zeiss AxioCam 305 Color camera (Carl Zeiss AG, Jena, Germany).