Whatman nucleopore track etch membranes

Mouse foregut regions (E8.5) or whole lungs (E11.5) were dissected from embryos in Hank’s balanced salt solution, explanted onto 8 μm pore Whatman Nucleopore Track-Etch Membranes (Millipore) and cultured in DMEM (Gibco) with 5% fetal bovine serum (FBS, Sigma) for lung explant culture or 20% FBS for foregut explant culture (Havrilak et al., 2017 (link); Havrilak and Shannon, 2015 (link)). Explants were cultured at 37°C in a 5% CO₂ incubator for 1-3 days. Whole-mount immunostaining was performed as previously described (Havrilak and Shannon, 2015 (link)). Images were captured on a Nikon A1Rsi inverted laser confocal microscope and composed using Bitplane Imaris software.

Timed pregnant mice were sacrificed on gestational day E8.5, and
individual embryos were staged according to somite number (ss). Experiments were
performed using embryos between 4–12ss. For E8.5 foregut cultures, the
neural headfolds and forebrain were usually removed, as well as tissue caudal to
the anterior intestinal portal (AIP). For foregut cultures in which liver and
pancreas formation were studied, embryos were cut 4 somites caudal to the AIP to
ensure that dorsal pancreas progenitors were captured within the region being
cultured (Angelo et al., 2012 (link)). Regions
containing the foregut were dissected in Hank's balanced salt solution
(HBSS), explanted onto 8 µm pore Whatman Nucleopore Track-Etch Membranes
(Millipore) or Transwell Collagen Filters (Costar) and cultured for 2–5
days in DMEM (Gibco) +20% fetal bovine serum (FBS; Sigma). In some
experiments 20% FBS was replaced with 20% KnockOut Serum
Replacement (KOSR; Gibco). The VEGF receptor inhibitors Ki8751 and SU5416 (both
from Calbiochem) were used at 10 µM. These compounds have been shown to
effectively inhibit VEGF signaling at this concentration (Fong et al., 1999 (link); Havrilak
and Shannon, 2015b; Kubo et al.,
2005). In all experiments DMSO served as a vehicle control.