Plan apo 20x

Samples were permeabilized and blocked in incubation buffer (0.25 % Triton X-100 and 1% bovine serum albumin in PBS) for 60 minutes. Samples were then treated with primary antibodies (see Table S1) in incubation buffer either at 4°C overnight or four hours at room temperature. Samples were rinsed with wash buffer (0.05% Triton X-100 in PBS; 2 x 60 minutes) and treated with incubation buffer at least 60 additional minutes at room temperature. Samples were treated with 1:200 secondary antibodies (Alexa-conjugated, Thermo Fisher Scientific, USA) and 5μg/mL DAPI (4′6-Diamidino-2-Phenylindole Dihydrochloride, MP Biomedicals, 157574) in incubation buffer overnight at 4°C or at least two hours at room temperature. Samples were mounted by adding a drop of Aqua Polymount solution (Polysciences, Inc.) to the well and placing a round glass coverslip over the top. The mounted samples were stored overnight at 4°C and then sealed around the edges with fingernail sealant until imaging.
Confocal immunofluorescence microscopy images were collected using a Nikon A1R laser scanning confocal microscope with Plan Apo 10x, Plan Fluor 20x Ph1 DLL, or Plan Apo 20x DIC M objectives. Images were processed using NIS Elements and ImageJ. Some z-stacks were aligned using the “Align Current ND Document” (NIS Elements) before creating maximum projection images.

Cells were seeded and cultured on Matrigel-coated glass-bottom culture dishes (MatTek, 12- or 24-well dishes) for differentiation. Cultured cells were then washed with PBS once then fixed with BD Cytofix at 4°C for 15 min. Cells were then permeabilized with 0.2% Triton X-100 (in PBS) at room temperature for 30 min. Cells were then blocked with blocking buffer (2%BSA and 1%FBS in PBS) for 1 h at room temperature followed by staining with primary antibody diluted in blocking buffer at 4°C overnight. The next day, the cells were washed three times with the blocking buffer before being incubated with AlexaFluor secondary antibodies (Thermo Fisher Scientific, 1:1000 dilutions in blocking buffer) and DAPI or Hoechst 33342 (Sigma, Cat. No. B2261) for 1 h at room temperature. Cells were then washed three times with the blocking buffer and ready for imaging. All the primary antibodies used in this study can be found in the Key Resources Table. Immunofluorescence images were collected using a Nikon A1R laser scanning confocal microscope with Plan Apo 10x, Plan Fluor 20x Ph1 DLL, or Plan Apo 20x DIC M objectives. Images were processed using NIS Elements or ImageJ. Z stacks are presented as maximum intensity projection images.