Histological Analysis of Mouse Embryos

The lower bodies of E18.5 mouse embryos were fixed with 4% paraformaldehyde (PFA) at 4 °C overnight. Fixed samples were further processed using a Tissue-Tek VIP-VI automatic infiltration processor (Sakura Finetek, Tokyo, Japan) with a standard 19 h program, followed by transfer into moulds. The samples were subsequently embedded with paraffin wax and sectioned perpendicularly against the head–tail axis using a microtome (RM2125RT, Leica, Wetzlar, Germany) set at a thickness of 4 μm. Sectioned tissues were mounted on New Silane II coated glass slides (Muto Pure Chemicals, Tokyo, Japan) and incubated on a paraffin extension plate (Leica) at 45 °C overnight. Post deparaffinization, slides were soaked in Mayer’s haematoxylin solution for 10 min followed by washing with water for 20 min. Slides were then incubated in 1% Eosin Y solution for 5 min before visualization under an all-in-one microscope BZ-9000 (Keyence, Osaka, Japan).