Anti e cadherin

Western blot assays were performed as previously described [24 (link)]. Antibodies used in the assays include: anti-HA, anti-phospho-p53 (Ser-15), anti-p53, anti-phospho-CHK2 (Thr-68), anti-phospho-CDK2 (Thr-160), anti-phospho-Rb (Ser-807/811), anti-Rb, anti-phospho-CDC2 (Tyr-15), anti-Smad2, anti-phospho-Smad2 (Ser465/467) (Cell Signaling Technology Inc.); anti-p21^WAF1 (Calbiochem Inc., Billerica, MA, USA); anti-CDK2, anti-CDC2 (Santa Cruz Biotechnology Inc.); anti-CDC6, anti-α-tubulin, anti-Vimentin, anti-E-cadherin (Life Technologies Inc.).

PHTs were cultured in standard conditions in the presence of PJ- or glucose-containing DMEM for 24 h and then fixed with methanol for 20 min at −20°C. After blocking for 1 h at room temperature with PBS with 5% bovine serum albumin (Sigma), fixed cells were incubated with either no primary antibody or a mixture of anti-NDRG1 (1:200; Catalog # 426200, Life Technologies) and anti-E-cadherin (1:200; Catalog # 18-0223, Life Technologies) antibodies overnight at 4°C. Cells were washed three times with PBS and incubated with a 1:500 dilution of DRAQ5 (Biostatus Unlimited, Leicestershire, UK) to stain DNA, and with appropriate secondary antibodies, as described [25 (link)]. One-micron-thick optical-section images were acquired by confocal microscopy using 600X total magnification and identical acquisition settings for all samples, as described [25 (link)]. The percent of cells expressing detectable NDRG1 was scored in five random fields, containing over 50 nuclei each, using trophoblasts from three different placentas, with the observer blinded to the treatment condition.

Paraffin-embedded specimens underwent hematoxylin and eosin (H&E) staining for histologic examination. To confirm a perceived increased density of goblet cells, we also performed period acid-Schiff (PAS) Alcian blue stains on selected samples. Immunofluorescence of OCT-embedded human tissue were challenging due to a rapid degradation and loss of immunofluorescence staining. To address this, tissues were embedded immediately in OCT compound and snap-frozen in liquid nitrogen. A modification of previous techniques allowed for an improvement in the detection of the immunofluorescence signal. Cryosections (8–10µm) were fixed, soaked in 2% formalin for 15 min, and washed three times with 1× Phosphate Buffered Saline (PBS) for 10 min. The samples were then incubated in blocking buffer (10% goat serum) for 1 h at room temperature, and incubated with primary antibodies diluted in 2% goat serum (mouse monoclonal anti-ZO-1 1:100, anti-occludin 0.6:100, anti-claudin-4 at 0.5:100 and anti-E-cadherin at 0.3:100 (Life Technologies, Invitrogen™, Carlsbad, CA) at 4°C overnight. After 3 additional 10 min washes in PBS, slides were incubated with corresponding secondary antibodies for 1 h and mounted with ProLong® Gold antifade reagent with 4’6-diamidino-2-phenylindole (DAPI) (Life Technologies). Slides were visualized using a Nikon A-1 Confocal fluorescence microscope.

In order to assess the formation of intercellular junctions, the trophoblast cells and HPVECs were fixed in 4% paraformaldehyde (PFA) for 15 minutes, permeabilized in 0.25% Triton X-100 for 10 minutes, and then incubated in 2% bovine serum albumin (BSA) for 1 hour. All steps were performed at room temperature. The trophoblast cells and HPVECs were incubated with anti-E-cadherin (Life Technologies) and anti-VE-cadherin antibodies (Cell Signaling Technologies), respectively. These primary antibodies were diluted in 2% BSA and incubated in the microdevice for 1 hour at room temperature. Next, the samples were thoroughly washed with PBS. Secondary antibodies (Life Technologies) were diluted in 2% BSA, incubated for 45 minutes at room temperature, and then washed with PBS. Nuclei were labeled using DAPI subsequent to the secondary antibody incubation. Following staining, the membrane was carefully removed from the microdevice and mounted onto a coverslip. Images were acquired using an inverted microscope (Zeiss Axio Observer) and a confocal laser-scanning microscope (Leica TCS SP8). Image processing and three-dimensional rendering were carried out using Volocity (PerkinElmer).

The following primary antibodies were used: anti-K14 (polyclonal
rabbit, 1:1000, Thermo Fisher Scientific), anti-GFP (chicken, 1:1000,
Abcam), anti-SOX2 (rabbit, 1:100, Abcam), anti-Vimentin (Rabbit, 1:200,
Abcam), Anti-ECadherin (rat, clone DECMA-1, 1:1000, eBioscience), anti-p63
(polyclonal rabbit, 1:200, Santa Cruz), Anti-Zeb1 (polyclonal rabbit, 1:300,
Bethyl), Anti-Zeb2 (polyclonal rabbit, 1:200, Sigma), anti-EpCam (rabbit
polyclonal, 1:200, Abcam). The following secondary antibodies were used:
anti-rabbit, anti-rat, anti-chicken, conjugated to AlexaFluor488 (1:400,
Molecular Probes), to rhodamine Red-X or to Cy5 (1:400, Jackson
ImmunoResearch).