Vecad

IHC was performed to detect the expression levels of different proteins. Tissue sections were deparaffinized in xylene and rehydrated by gradient alcohol prior to IHC. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in methanol for 30 min. The tissue sections were heated using 0.01 M citric acid buffer for 10 min in a microwave oven. The slides were stained with antibodies against Twist1 (1:100, Santa Cruz Biotechnology), TP (1:100, Abcam), VE–cad (1:50, Abcam), VEGFR1 (1:250, Abcam), VEGFR2 (1:50, Abcam), or CD31 (1:50, Abcam). After incubation with horseradish peroxidase-conjugated goat anti-rabbit/mouse immunoglobulin G (IgG), the sections were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin or periodic acid–Schiff (PAS). Finally, the sections were dehydrated and mounted. IHC staining was scored by multiplying the positive degree (0 for none, 1 for weak brown, 2 for moderate brown, and 3 for strong brown) and positive rate (1 for 0–25%, 2 for 25%–50%, 3 for 50%–75%, and 4 for 75%–100%). Sections with staining indices >6 were classified to the high-expression group. PAS-positive channels without CD31 staining were classified to the VM group. Five random ×40 fields per HCC tissue were scored with morphology consistent with mimicry. All quantification experiments were performed in a blinded setting.

Prior to immunofluorescence staining, nonspecific binding was blocked with 2% bovine serum albumin (BSA)/0.5% Triton X-100 for 1 h. Staining for CD31 (Abcam), VE-cadherin (VE-cad; Abcam), von Willebrand Factor (vWF; Abcam), and α-smooth muscle actin (αSMA; Thermo Fisher Scientific) was accomplished through perfusion of immunofluorescence reagents through the microvessel network as described previously [53 (link)]. Secondary antibodies with fluorochromes Alexa Fluor 488, 567, or 647 were used. Vessels were imaged on a Nikon A1R confocal microscope.

Chick and mouse hearts and mouse embryos were fixed in 4% paraformaldehyde for overnight, washed with TBS, embedded with paraffin, and sectioned. Sections were deparaffinized and rehydrated. Antigen retrieval was completed using citrate buffer at pH 6.0. Samples were then washed with TBS, permeabilized with 0.3% Triton-X 100 in TBS, and blocked with 3% BSA, 20 mM MgCl, 0.3% Tween 20, 0.3 M Glycine, and 5% Donkey serum in 1xTBS. Samples were then incubated with the primary antibodies at 1:100 dilution with the blocking solution overnight. Primary antibodies used include YAP (mouse DSHB YAP1 8J19, mouse Santa Cruz sc-101199, rabbit cell signaling #14074), Lef1 (rabbit Cell Signaling #2230), pHH3 (rabbit Cell Signaling #9701), VE-cad (abcam ab33168), MF-20 (mouse DSHB MF 20), IB4 (Vector B-1205-.5), Phalloidin (Invitrogen A12379). Samples were washed with 0.3% Triton-X 100 in TBS and incubated with secondary antibodies at 1:100 dilution with 5% BSA in TBS at room temperature for 1 hr. Secondary antibodies used include species-specific Alexa Fluor 568 or 647. Samples were then washed again for 3x10 min with TBS and stained with DAPI. Images were taken with Zeiss LSM 710 confocal microscope.