Dapi pbs solution

Immunohistochemistry (IHC) was carried out on sections post-in situ hybridization in a 48-well plate beginning with three 30-minute PBS-T (1% Triton X-100) washes followed by incubation for one hour in PBS-T containing 10% fetal bovine serum (FBS). Primary antibodies as listed in S1 Table were applied overnight at 4° (Laminin IHC was performed on sections not previously processed by in situ hybridization). The following day, sections were washed five times for one hour each in PBS-T before incubation with secondary antibodies as listed in S1 Table overnight at 4°, rinsed five times in PBS-T, incubated with 200 ng/mL DAPI/PBS solution (Sigma) for 30 minutes, and mounted. For F-actin staining, sections that had not been processed by in situ hybridization were washed and blocked as above and incubated with Phalloidin-Alexa Fluor 488 (Molecular Probes A12379, 1:100 in DAPI/PBS solution) for 30 minutes. Images and Z-stacks were taken with a Zeiss 700 confocal microscope using a 63X oil objective. For Z-stacks, slices were imaged at 1 μm intervals and stacks were reconstructed to represent 1–3 μm projected images using ImageJ. The luminal area of the posterior cardinal veins was measured using ImageJ.

The fibroblasts seeded on cPU membranes were fixed in 4% paraformaldehyde for 10 min at room temperature, washed with PBS thrice for 5 min each time, soaked in 0.2% Triton X-100 (Beijing Solarbio Science & Technology, China) for 20 min, and rinsed in PBS thrice for 10 min each time. After that, the samples were blocked in 10% goat serum/PBS for 20 min at 37°C followed by incubation in the mouse anti-vimentin primary antibody (1 : 200 dilution in PBS) at 4°C overnight. After rinsing with PBS, the samples were incubated in FITC-conjugated goat anti-mouse IgG (1 : 50 dilution in PBS) for 2 h at 37°C in the dark room. Finally, after washing with PBS, the samples were dipped in a 4,6-diamidino-2-phenylindole dihydrochloride (DAPI)/PBS solution (Sigma, 3 mg/mL) for 5 min to stain the nuclei (blue fluorescence). The cells were observed under confocal laser scanning microscopy (CLSM, Olympus Fluoview-1000).