Jung frigocut cryostat

Dissected guts were washed in 1 × phosphate-buffered saline (PBS), cut into anterior, distal and middle portions and were fixed in 4% PFA at 4ºC, overnight (ON). Then, they were washed in cold PBS and immersed in 30% sucrose in PBS, ON at 4ºC, for cryopreservation. Individual guts were then embedded in mounting media – optimal cutting temperature compound (OCT, VWR International), snap-frozen in dry ice, and stored at -20ºC until cryosectioning. Cryosections were sliced at a 13 μm thickness using a Leica Jung Frigocut cryostat or a Leica CM1860 cryostat. Sections were air dried for 2 h then used for immunohistochemistry or frozen for up to 3 months at -20ºC before use.

Alkaline phosphatase wholemount in situ hybridisation experiments were performed using standard methods as described previously^{71 (link)}. Detailed protocols are available upon request. EC dCas9 expression was detected using Cas9 in situ probe⁷² . Immunohistochemistry to detect pERK was performed using Phospho-p44/42 Erk1.2 (Thr202.Tyr204) Rabbit mAb (#4370, cell signal, 1:250) and quantified by normalising EC signal against ERK staining within the neural tube as described^{7 (link),73 (link)}. Immunohistochemistry to detect dCas9 was performed on whole-mount and sectioned embryos as described^{74 (link)} using mouse anti-CRISPR/Cas9 (7A9-3A3) (Novus Biologicals, NBP2-36440, 1:100), chicken anti-GFP (Abcam, ab13970, 1:500) primary antibodies, goat anti-mouse IgG (H&L) Alexa Fluor® 647 (Thermo Fisher A21235, 1:1000), and goat anti-chicken IgY (H&L) Alexa Fluor® 488 (Thermo Fisher A11039, 1:1000) secondary antibodies. Sectioned embryos were fixed at 3 dpf in 4% paraformaldehyde overnight, washed into 30% sucrose and sectioned at 14 μm thickness on a Jung Frigocut cryostat (Leica).