Bp165 25

Images were acquired by lightsheet microscopy, as described previously^{43 (link)}. Brielfy, specimens were warmed and transferred to 2% low melting point agarose (Fisher BP165–25) in PBS at 37°C, then embedded in glass capillary tubes with paired pistons (Sigma Z328510 paired with BR701938, or Sigma Z328502 paired with BR701934) for embryos, or tip-truncated 1mL syringes (Becton Dickinson) for hearts. After the gel solidified, the capillaries or syringes were suspended specimen-down from 14mL polystyrene tubes sealed with Parafilm. Columns containing the specimen were partially extended into an ample volume optical clearing solution (OCS, EasyIndex EI-Z1001, LifeCanvas) overnight. With immersion in OCS, specimens were rotated and imaged from multi-view whole-volume approach on a Light Sheet Z.1 microscope (Carl Zeiss Imaging). One of two objective setups was used: EC Plan Neofluar 5X/0.16 with 5X/0.1 pair (hearts), or Clr Plan Neofluar 20X/1.0 paired with 10X/0.2 clearing pair (embryos). Z-stacks were collected at each view angle at the optimal slice thickness determined by Zeiss’ Zen software, ranging from 1.42 to 4.95 μm.

Images were acquired by lightsheet microscopy, as described previously ^{37 (link)}. Brielfy, specimens were warmed and transferred to 2% low melting point agarose (Fisher BP165-25) in PBS at 37°C, then embedded in glass capillary tubes with paired pistons (Sigma Z328510 paired with BR701938, or Sigma Z328502 paired with BR701934) for embryos, or tip-truncated 1mL syringes (Becton Dickinson) for hearts. After the gel solidified, the capillaries or syringes were suspended specimen-down from 14mL polystyrene tubes sealed with Parafilm. Columns containing the specimen were partially extended into an ample volume optical clearing solution (OCS, EasyIndex EI-Z1001, LifeCanvas). Following overnight incubation in OCS, specimen capillaries were retracted and were brought to a Light Sheet Z.1 microscope coupled with ZEN software (Carl Zeiss Imaging). With immersion in OCS, specimens were rotated and imaged from multi-view whole-volume approach, to improve fluorescence signal intensity and resolution captured throughout the entire heart volume. One of two objective setups was used: EC Plan Neofluar 5X/0.16 with 5X/0.1 pair (hearts), or Clr Plan Neofluar 20X/1.0 paired with 10X/0.2 clearing pair (embryos). Z-stacks were collected at each view angle at the optimal slice thickness determined by Zeiss' ZEN software, ranging from 1.42 to 4.95 µm.

The ability of cancer cells to form colonies was characterized using a soft agar assay. This assay required 21 days of growth on the soft agar medium. At the end of 3 weeks, a number of colonies formed per petri dish were counted using a crystal violet stain. Briefly, 1% sterile agar solution was warmed in a microwave and place to 37 °C water bath to cool down. 500 μg of agarose powder (BP165-25, Thermo Fisher Scientific, Fair Lawn, New Jersey, USA) was dissolved in 50 mL distilled water. The bottom of the petri dish (502014-07P, Sterilin petri dishes 9.6 cm², Dynalon Labware, Rochester, NY, USA) was coated with 0.7% agar and 0.3% CM by adding 3 ml/dish at room temperature for 30 min. following this, the upper layer of 3 mL of agar solution with 0.3% agar and 0.7% cell suspension (3125 cells/cm²) was plated. The top agar layer was allowed to solidify and then incubated for 3 weeks at 37 °C in 5% CO₂. CM was refreshed 2 times a week. In 21 days crystal violet was used as a staining for colonies (C3886, Sigma-Aldrich, Munich, Germany) and counting was performed using Leica DMI3000 B light microscope.

Stained embryos were embedded into low-melting agarose (BP165-25; Thermo Fisher) containing 0.1 μm fluorescent beads (F8801; Thermo Fisher). The embedded embryos were then imaged in a Zeiss Light-sheet Z1 microscope under ×20 water objective from four angles. The resulting multi-view images were registered using ImageJ plugin multi-view reconstruction.