Cryostar hm560

Isolated mouse embryos were fixed for 3 h or overnight with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). After washed with PBS, the embryos were incubated with 10%, 20%, and 30% sucrose in PBS and embedded with O.C.T. compound (Sakura Finetek Japan) at –80 °C. Coronal sections of embedded embryos were prepared by Cryostar HM560 or NX70 (Thermo Fisher).

Lungs were harvested, embedded in optimal cutting temperature media, and snap-frozen in liquid nitrogen. Then, 10 μm thickness sections were made using a cryostat (Cryo-Star HM 560, Thermo Scientific, Walldorf, Germany). The sections were fixed with paraformaldehyde (PFA) 4%, and unspecific labeling was blocked with HBSS supplemented with 1% Fc block and 10% fetal calf serum. Primary anti-CD31 (10 μg/mL, R&D Systems, Minneapolis, MN, USA) and anti-HS (10 μg/mL, Amsbio, Cambridge, MA, USA; clone F58-10E4) antibodies were added overnight at 4 °C. Subsequently, the sections were washed with HBSS, and the secondary antibodies were added for 2 h. Images were taken using a 25× objective in a Dragonfly spinning-disk confocal microscope (Andor Technology, Concord, MA, USA). The pictures were analyzed using FIJI software.