Immunofluorescence Imaging of Gastruloids and Spheroids

To perform immunofluorescence on gastruloids and spheroids we used the protocol previously described [21 (link),22 ]. Briefly, gastruloids were washed (3×, 10 min) at RT with PBS, then with PBS/10% FBS/0.5% Triton X-PBSFT (3×, 10 min) and finally with PBSFT (1 h, 4 °C). Gastruloids and spheroids were then incubated with the following specific antibodies (48 h, at 4 °C) on a low-speed orbital rocker: E-cadherin (1:250, Takara, Saint-Germain-en-Laye, France); Oct4 (1:100, Santa Cruz Biotechnology, Dallas, TX, USA); Nanog (1:400, Cell Signaling, Danvers, MA, USA); Cdx2 (1:100, Cell Signaling, Danvers, MA, USA); Bra (1:500, Cell Signaling, Danvers, MA, USA) and Sox2 (1:100, Cell Signaling, Danvers, MA, USA). Images were obtained using a confocal Nikon A1 microscope. Then NIS Element C (Nikon, Tokyo, Japan) software was used for image acquisition/elaboration.