Mouse anti ve cadherin

For uterine analyses, 5 μm transverse sections through the non-pregnant and E3.5 uteri were generated. For implantation site analyses, frontal sections through uterus/implantation site were generated for E6.5 at 7 μm and for E7.5 with VEGFR-1 blockade at 12 μm. Implantation was confirmed by H&E staining every 5th section. Specific staining was performed at least 3 times and 5 different uterine sections or implantation sites were analyzed.
Sections were stained as previously described [27 (link)]. Primary antibodies included anti-mouse VEGFR-1 (R&D Systems, AF471), anti-mouse F4/80 (eBioscience, 14–4801), anti-mouse CD31 (BD Biosciences, 553370), anti-mouse endomucin (Santa Cruz, sc-65495), anti-mouse VE-cadherin (BD Biosciences, 550548), and anti-mouse CD11b (Abcam ab8878). For IHC staining, biotin rabbit anti-goat IgG (Vector, BA-5000), biotin goat anti-rat IgG (BD Biosciences, 559286), the avidin/biotin blocking kit (Vector, SP-2001), the Vectastain ABC kit and DAB substrate kit (Vector, SK-4100) were used. Sections were counterstained with hematoxylin. For IF experiments, the following secondary antibodies were used: donkey anti goat-IgG Alexa-Fluor 594 (Invitrogen, A11058) and donkey anti rat-IgG Alexa-Fluor 488 (Invitrogen, A2108). Slides were covered with Vectashield containing with 4′, 6-diamidino-2-phenylindole (Vector, H-1200) for nuclear visualization.

Lungs from P7 Wt1^LoxP/LoxP; UBC-CreERT2 mice were digested with type 1 collagenase (Worthington; 1 mg/ml) for 1 h at 37°C, with mild shaking every 2-5 min. The collagenase was inactivated by adding DMEM containing 10% of inactivated FBS. The cell suspensions were filtrated through a 100 µm cell strainer and pelleted by centrifugation (5 min, 1200 rpm). The dissociated cells were washed twice with PBS/BSA 0.5% followed by positive selection with anti-mouse VE-cadherin (Pharmingen, 555289) coated with magnetic beads for 30 min (Invitrogen, 11035). Purified cells were seeded on plates coated with gelatin plates (0.5%) in complete Endothelial Cell Growth Medium 2 (EGM-2; which is EBM-2 medium supplemented with EGM-2 SingleQuots). Confluent ECs were re-purified with anti-mouse VE-cadherin-coated magnetic beads. To induce Wt1 deletion, 4-hydroxytamoxifen (4-OHT, 1 µM, Sigma-Aldrich, H7904) or vehicle (ethanol) was added to the culture medium. All the experiments were performed 72 h after the addition of 4-OHT or vehicle, and in ECs obtained immediately after the second round of purification.

The following antibodies were used for western blotting: rat anti-HA (3F10; Roche/Sigma-Aldrich), rabbit anti-myc, mouse anti-RhoA, rabbit anti-MLC2 (Santa Cruz Technology), rabbit anti-FAM40B (Sigma-Aldrich), rabbit anti-pThr18/pSer19-MLC2 (Cell Signaling), mouse anti-GADPH (Millipore), secondary HRP-conjugated sheep anti-mouse IgG and donkey anti-rabbit IgG (GE Healthcare). Antibodies for immunofluorescence analysis were: rabbit anti-HA (Santa Cruz Technology), rabbit anti-pSer19-MLC2 (Cell Signaling), mouse anti-VE-cadherin (BD Biosciences), rabbit anti-ZO-1 (#61–7300; ThermoFisher Scientific), secondary AlexaFluor-488 and -647-conjugated antibodies (Molecular Probes). Cells were also stained with DAPI (DNA) and AlexaFluor-546-labeled phalloidin (F-actin; Molecular Probes).