Freshly dissected embryos were fixed in X-gal buffer containing 0.2% glutaraldehyde and 1% formaldehyde on ice for 15 min and subjected to modifications from the previous protocol (Cui et al. 2020 (link), Tremblay et al. 2000 (link)). In brief, the fixed embryos were washed with X-gal buffer (PBS, 5 mM EGTA, 2 mM MgCl:6H2O, 0.2% NP-40, 0.2 mM deoxycholate) for 10 min three times, and stained with X-gal stain (X-gal buffer with 5mM potassium ferricyanide, 5mM potassium ferrocyanide, and 0.5 mg/ml X-gal) for 48 hours at 37°C. Subsequently, embryos were dehydrated in ethanol, cleared in xylene, embedded in paraffin, and sectioned at 7 μm. Slides were deparaffinated in xylene, counterstained with Eosin and coverslipped in Cytoseal (Thermo Fisher Scientific, Waltham, MA, USA) and imaged with a Pannoramic MIDI II slide scanner (3DHISTECH Ltd., Budapest, Hungary).