Goat anti mouse igg af488

The following commercially available antibodies were used at the indicated concentrations for western blot: Anti‐ZFP207 (Santa Cruz Biotechnology, sc‐271943, 1:500), Anti–β‐ACTIN (Sigma‐Aldrich, A5441, 1:2,500), Anti‐OCT3/4 (Santa Cruz Biotechnology, sc8628, 1:2,500), Anti‐Nanog (Santa Cruz Biotechnology, sc‐374001, 1:1,000), Anti‐Sox2 (Santa Cruz Biotechnology, sc‐398254, 1:1,000), Anti‐SFRS11 antibody (Abcam, ab196801, 1:2,000), Goat Anti‐Rabbit IgG H&L (HRP) (Abcam, ab6721, 1:5,000), Goat Anti‐Mouse IgG H&L (HRP) (Abcam, 1:1,000, ab6789), Rabbit Anti‐Goat IgG H&L (HRP) (Abcam, 1:5,000, ab6741), and Rabbit Anti‐ goat IgG (HRP) (Abcam, ab6771, 1:5,000), Anti‐Flag (Sigma, F3165, 1:1,000). In all western blots, β‐ACTIN was used as a loading control (Sigma, A5441, 1:2,500). For IF staining, we used Anti‐SSEA1 (Invitrogen, MA5‐17042, 1:250), Anti‐Nestin (Abcam, ab81462, 1:50), Anti‐Tuj1 (Abcam, ab18207, 1:200), Anti‐Caspase 3 Antibody, active (cleaved) form (MERK, AB3623, 1:100), Goat anti‐mouse IgG AF488 (Invitrogen, A11029, 1:1,000), Goat anti‐rabbit IgG AF568 (Invitrogen, A11011, 1:1,000), Anti‐Mouse IgG HRP (Abcam, ab6789, 1:1,000), and Donkey Anti‐Goat IgG AF594 (Invitrogen, A11058, 1:250).

iMACs were permeabilized with methanol for 20 min at -20°C. Afterwards, cells were incubated for 1h in 0.1% Triton X100 and 2% BSA (Sigma Aldrich) diluted in PBS. This was followed by overnight incubation at 4°C with 1:200 anti- LAMP-1 (D401sm #15665, Life Technologies). The next day, the iMACs were washed 3 x 5 min with PBS and incubated for 1h with 1:1000 goat anti-mouse IgG-AF488 (A11001, Invitrogen). Afterwards the cells were washed 3 x 5 min with PBS again. The wells were filled up with PBS and the plates were stored at 4°C.

The following procedure was adapted from our previous work^{12 (link)}. Unless stated, about 10–20 organoids were collected into 1.7 ml Eppendorf tubes (Corning Inc). After aspirating the media, the organoids were fixed in 4% formaldehyde (Polysciences Inc, Warrington, PA) for 15 minutes at 4 °C and were washed 3 times with cold PBS. The organoids were permeabilized with 0.1% Tween-20 in PBS for 10 minutes at 4 °C and washed 3 times. Organoids were exposed to Protein Block (Dako Group, Troy, MI) for 1 hr at RT, and organoids incubated at 4 °C overnight in Antibody Diluent (Dako) solution containing primary antibodies, anti-beta Catenin (1:500, Abcam), anti-ZO-1 (1:1000, Millipore), and anti-Claudin-5 (1:500, Millipore). Organoids were subsequently washed 3 times and incubated with AF488 Goat anti-Mouse IgG (1:1000, Life Technologies), AF594 Goat Anti-Rabbit IgG (1:1000, Life Technologies) in Antibody Diluent (Dako) overnight at 4^oC. Nuclear staining was performed by incubating the organoids with DAPI (1:1000) in PBS for 10 minutes. The organoids were washed and were imaged using the Olympus Fluoview Fv10i (Olympus) laser scanning confocal microscope. Unless stated, at least three randomly selected organoids were imaged for each stain.

Spheroids were transferred to 1.7 mL Eppendorf tubes, fixed in 4% formaldehyde (Polysciences Inc, Warrington, PA) for 30 min at 4 °C, and washed three times with cold PBS. The spheroids were then permeabilized with 0.1% Tween-20 in phosphate buffered saline (PBS) for 10 min at TR and washed three times with cold PBS. Then, they were treated with Protein Block (Dako Group, Troy, MI) for 2 h at room temperature. Then, they were incubated at 4 °C overnight in Antibody Diluent (Dako) solution which contained the primary antibodies anti-ZO-1 and anti-CD31 (1:200, Corning). The spheroids were subsequently washed three times with cold PBS and incubated with AF488 goat-anti-mouse IgG (1:500, Life Technologies) overnight in Antibody Diluent (Dako) at 4 °C. Nuclear staining was performed by incubation of the spheroids with DAPI (1:1000) in PBS for 10 min. The spheroids were washed and imaged using CLSM. For imaging, at least randomly selected spheroids were randomly selected for each stain.

Antibodies were purchased from the following sources: anti-CD99 FITC conjugated (BD Biosciences, catalog no: 555688, concentration 1:20), anti-FLI1 (Abcam, catalog no: 133485, concentration 1:2,000), anti-BARD1 (Bethyl Laboratories, catalog no: A300–263A, 1:2,000), anti-BARD1 (Abcam, catalog no: ab50984, concentration 1:100), anti-GBP1 (Abcam, catalog no: 131255, concentration 1:300 for IHC), anti-GBP1 (Santa Cruz Biotechnology, catalog no: sc-53857, concentration 1:200 for Western blot analysis), anti-phospho (Ser 139)-γH2A.X (Millipore Sigma, catalog no: 05–636, concentration 1:2,500 for immunofluorescence), anti-phospho (Ser 139)-γH2A.X (Invitrogen, catalog no: MA1–2022, concentration 1:1,000 for Western blot analysis), goat anti-mouse IgG AF-488 (Thermo Fisher Scientific, catalog no: A-11001, concentration 1:2,000), tubulin (Cell Signaling Technology, catalog no: 2144S, concentration 1:5,000), vinculin (Cell Signaling Technology, clone E1E9V, catalog no: 13901S, concentration 1:5,000), and anti-rabbit IgG-horseradish peroxidase (HRP) (Promega, catalog no: W401B). Additional specialized reagents include: BMN 673 (talazoparib; Cayman Chemical, catalog no: 19782), MK-4827 tosylate (niraparib; Cayman Chemical, catalog no: 20842), DMSO (MP Biomedicals, catalog no: 196055), doxorubicin (Cayman Chemical, catalog no: 15007), and IFNγ (R&D systems, cat no: 285IF100).

To determine if coagulation proteins are present in the cytoplasm of LSECs, GMVECs, and HUVECs, cells were grown on glass coverslips (1.5-mm thick) pre-coated with gelatin and were washed with PBS 3X at each step. Washed cells were fixed with 1% p-formaldehyde in PBS and followed by 0.02% Triton-X to enable intracellular staining. The cells were stained for 15 min with each primary (diluted 1:100 in PBS containing 1% BSA) plus fluorescent AF-labeled secondary antibody at 20 µg/ml (Life Technologies). Primary antibodies purchased through Haematologic Technologies include: sheep anti-human FIX (PAHFIX-SAP), sheep anti-human FX (PAHFX-S), sheep anti-human FVII (PFVII-S), and sheep anti-human prothrombin (PAHFII-SAP) plus secondary donkey anti-sheep IgG-AF-647. Additional primary antibodies that were used are: rabbit anti-human VWF (Ramco Laboratories) plus chicken anti-rabbit IgG-AF-488; and mouse anti-human FVIII (ThermoFisher, F8-5.5.72) plus goat anti-mouse IgG-AF-488. Cell nuclei were detected with DAPI (4′,6-diamidino-2-phenylindole, 1.5 µg/ml) that was included in the mounting medium (Fluoro-Gel II, Electron Microscopy Sciences).