Axio observer z1 microscope

Cells were washed twice with DPBS and fixed for either 20 min in 4% paraformaldehyde (Sigma-Aldrich) or 10 min in 100% ice-cold methanol. Cells were washed 3 times with DPBS and blocked for a minimum of 1 h in PBS or TBS containing 5% donkey serum and 0.3% Triton X-100 (PBS-DT and TBS-DT, respectively). Cells were incubated with primary antibody diluted in PBS or TBS containing 5% donkey serum (PBSD and TBSD, respectively) or in PBS-DT or TBS-DT overnight at 4 °C. Following primary antibody incubation (see Additional file 1: Table S1), cells were rinsed once with PBS or TBS and washed five times with PBS or TBS for a minimum of 5 min per wash. Cells were incubated in secondary antibody (see Additional file 1: Table S2) diluted in the same buffer as primary antibody for a minimum of 1 h. Following secondary antibody incubation, cells were incubated with 4′,6-diamidino-2-pheny-lindoldihydrochloride (DAPI; Thermo Fisher Scientific) for 10 min to label nuclei. Cells were rinsed once and washed five times with PBS/TBS and then visualized using a Zeiss AxioObserver Z1 microscope or a Leica DMi8 microscope. An average of three images were taken for each stain and the entire field was visually assessed to ensure that the presented images are representative of the entire dish.

Xenopus laevis embryos were obtained as previously described (Afouda et al., 2005 (link)). Embryos and explants culture as well as embryos injection were as described in Afouda et al. (2008) (link) and Afouda and Hoppler (2011) (link). Animal cap explants were excised as previously described (Afouda, 2012 (link)) and where applicable dexamethasone was added at final concentration of 10 µM (at either stage 8 or 13, see Figures legends) for activation of GR-fusion proteins. Live GFP beating explants imaging was performed using ZEISS Axio Observer Z.1 microscope with the Axiovision software and movies were taken using Leica M60 microscope mounted with Leica MC 170 HD camera. Transgenic embryos were obtained from myl2-GFP lines. They were sourced from National Xenopus Resource (at Marine Biology Laboratory, Woods Hole, Massachusetts, USA) and were previously generated (Latinkic et al., 2004 (link)).

Fluorescence imaging was performed on a Zeiss LSM510 META confocal microscope, a Zeiss Axio Observer Z1 microscope with AxioCamMR3 camera, and a Leica DMI6000 with DFC365FX camera. The images were acquired and generated using Zen software version 3.1 (Carl Zeiss) and LAS AF Lite software version 4.0 (Leica), respectively. Hematoxylin and eosin whole slide images were acquired using a NanoZoomer- SQ digital slide scanner C13140-01 (Hamamatsu) and images were generated using NDP.view2 software version 2.9.29 (Hamamatsu).