Fluoro gel 2

Cells were fixed with 4% paraformaldehyde for 15 min, washed twice with PBS pH 7.4 for 5 min and incubated with blocking solution (10% normal donkey serum in 1X PBS with 0.3% Triton) for 1 h at room temperature. Cells were incubated with the primary antibodies TDP-43, ASRGL1 and NeuN (Supplementary Table 4) at 1:500 dilution in PBS with 1% donkey serum at 4 °C overnight. Then, cells were washed 3 times in PBS for 5 min each and incubated with the secondary antibodies (Thermo Fisher) at 1:500 in PBS with 1% donkey serum for an hour. After 3 washes with PBS, cells were mounted with Fluorogel II with DAPI mounting media (Electron microscopy Sciences). Slides were imaged using an LSM 510 META laser scanning confocal microscope (Carl Zeiss). Cells incubated with secondary antibody only were used as controls.

Upon ending the experiment, anterior segments were removed from the chambers, sectioned into quadrants and fixed in 4% paraformaldehyde/PBS for 6 h at 4 °C. Specimens were then immersed in 10% sucrose/PBS for 6 h, transferred to 30% sucrose overnight at 4 °C, and small wedges embedded in optimum cutting temperature compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA). Meridional 10 µm cryosections were dried at room temperature, rehydrated with DPBS Ca/Mg (Gibco/Life Technologies/ThermoFisher, cat #14040133) and blocked with 10% goat serum/PBS for 1 h. Subsequently, sections were incubated with a chicken anti-GFP polyclonal antibody (Aves Laboratories, Tigard, Oregon, USA cat #GFP-1020) (1:500) overnight at 4 °C followed by incubation with goat anti-chicken Alexa Fluor 594 (Invitrogen/ThermoFisher cat #A11042) (1:500) at room temperature for 2 h. All antibody solutions were made in 1% goat serum/0.3% Triton X-100 in PBS. After DPBS Ca/Mg washes, slides were mounted with DAPI-containing Fluoro-Gel II (Electron Microscopy Science, Hatfield, PA, USA cat #17985-50). No primary antibody controls were run in parallel. Images were captured on the IX71 Olympus microscope, DP80 monochrome camera and cellSens software described above.

Cells were plated onto glass coverslips (Fisher Scientific, Hampton, NH) placed in a 12‐well plate, fixed in 2% paraformaldehyde (Santa Cruz Biotechnology) for 20 min at RT.¹⁶ For anti‐HA staining, cells were permeabilized with 0.2% saponin/PBS for 30 min at RT. Cells were incubated with the following primary antibodies for 1 h at RT: anti‐HA mouse monoclonal (1:500); anti‐CD148 mouse monoclonal (1:500). Then, cells were incubated with Alexa Fluor 546 goat anti‐mouse IgG (1:500) for 30 min at RT. Nuclear staining was carried out with 0.01 mM DRAQ5 (Thermo Fisher Scientific) according to the manufacture's protocol. Coverslips were mounted on glass slides with Fluoro‐Gel II (Electron Microscopy Sciences, Hatfield, PA). Confocal images were acquired using the Zeiss 510 confocal microscope (Carl Zeiss, Jena, Germany).

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).

Cells grown on gelatin-coated glass coverslips (1-mm thick) were washed with PBS, fixed with 1% p-formaldehyde in PBS, and then treated with 0.02% Triton-X to allow internal staining. The cells were stained for 15 min with the relevant primary and fluorescent antibody pairs. The cell nuclei were detected with 1.5 μg/ml 4’,6-diamidino-2-phenylindole (DAPI) included in the mounting medium (Fluoro-Gel II, Electron Microscopy Sciences). In experiments where only cell surfaces were detected with antibodies, the treatment with Triton-X was omitted.