V2111

Embryos were imaged on a Leica M205 FCA stereoscope with a DFC7000T camera under reflected light darkfield. For each embryo, images were taken at multiple focal distances. These images were then focus-stacked using Helicon Focus or Photoshop to produce a single image with all cells in focus. To prevent movement between imaging different focal planes, post-hatching embryos were anaesthetized by submersion in 0.02% Tricaine methanesulphonate (Sigma-Aldrich E10521) for the duration of imaging (approx. 2 min per embryo) with the yolk supported in a shallow well of solidified 1% low-melting agarose (Promega, V2111). Adult fish were photographed using a Panasonic DMC GX7 camera with a Panasonic Lumix G 20 mm pancake lens, in a photography tank containing a scale. Lighting conditions were standardized using two light sources, one either side of the camera and a grey background.

Immunostained slices were placed flat in a glass petri dish and covered with a 2% solution of analytical grade low-melting point agarose (V2111; Promega, Fitchburg, WI) in water. Once set, slices were subjected to a serial dehydration protocol; 2 h in 50% EtOH in water, 2 h in 70% EtOH in water, 2 h in 100% EtOH and 24 h in 100% EtOH. All EtOH was then removed, and cyanoacrylate super glue was used to adhere the edges of the agarose disc to the petri dish prior to clearing with ethyl cinnamate (1 12,372; Sigma-Aldrich). The agarose disc and embedded tissue became clear within 3 h.

Prior to imaging, gastruloids were embedded in agarose and cleared with RIMS [Refractive Index Matching Solution; 133% w/v Histodenz (Sigma-Aldrich, D2158) in 0.02 M PB (see below)]. To this end, 10% low melting point (LMP), analytical grade (Promega, V2111) agarose was prepared in PBS, incubated at 80°C for 15 min, and cooled down to 37°C for 3 min in a thermomixer. Samples were washed twice with PBS for 10 min, post-fixed in 4% PFA for 20 min, and washed three times with 0.1 M phosphate buffer (PB; 0.025 M NaH₂PO₄, 0.075 M Na₂HPO₄, pH 7.4). The pipette was set to 20 µl and the gastruloids were stabilized on the ibidi plate with a drop of LMP agarose for 5 min until the agarose was dry. Clearing was performed by incubation in 200 µl RIMS on a rocking platform at 4°C for one to several days. Cleared gastruloids were imaged with a ZEISS LSM 880 Airyscan in confocal, Airyscan or Fastairy mode, using a Plan-Apochromat 20×/NA=0.8 objective, lateral pixel size of 0.1.2-1.2 µm and a typical z-spacing ranging from 1.9 to 3.3 μm and appropriate laser/filters for DAPI, Alexa Fluor 546 and Alexa Fluor 633 or combinations thereof. Brightfield images of non-cleared gastruloids were acquired with a ZEISS Celldiscoverer 7, with a Plan-Apochromat 5×/NA=0.3 objective and a 1× post-magnification, lateral pixel size of 0.9 µm and a typical z-spacing of 10 μm, running under ZEN Blue v3.1.

Organoids were washed with PBS and incubated for 30 min at 37 °C with 300 µl of low melting agarose 2% (Promega, V2111). Polymerized agarose was transferred to a 1.5 ml Eppendorf and fixed in 4% paraformaldehyde overnight at 4 °C under agitation. The sample was then washed three times in PBS 1× and put in PBS sucrose 30% overnight at 4 °C. Finally, organoids were incubated with gelatin 7.5%/sucrose 15% in PBS at 37 °C for at least 30 min and put in mold to freeze at −80 °C.