Anti cd34

Protein in HUVECs was extracted with RIPA buffer containing PMSF (Solarbio, Beijing, China). The supernatant of the lysis and exosome derived from HCT116 and SW480 was quantified with a BCA kit. Then, more details are provided below [11 (link)]. The following antibodies were used: anti-CD9 (Abcam, ab263019); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD34 (Proteintech, 14,486–1-AP); anti-Integrin β1 (Proteintech, 12,594–1-AP); anti-VEGFA (Proteintech,19,003–1-AP); anti-PDK2 (Abcepta, AP7039b); anti-Akt (Abcam, ab179463); anti-p-Akt (Ser473); anti-GAPDH (Proteintech, 60,004–1-Ig). IF and IHC was performed as previously described [11 (link)]. IF was performed using anti-CD34 (Proteintech, 14,486–1-AP). Then, the image of IF was obtained using the LSM880 confocal microscope system (Zeiss, Jena, Germany). IHC was performed using anti-CD31 (Proteintech, 11,265–1-AP) and anti-CD34 (Proteintech, 14,486–1-AP). The images of IHC were acquired using a fluorescence microscope system (Olympus, Tokyo, Japan).

Tissue-embedded sections were washed repeatedly in xylene (three times), 100% ethanol (two times), 70% ethanol (two times) and water (two times) for 10 min. The sections were then boiled in sodium citrate buffer for 1 h. The peroxidase blocker was added drop wise for 10 min, and tissues were incubated with the primary antibody at 4 °C overnight. The primary antibodies used were anti-CD34 (cat:14486-1-AP, Proteintech, China, 1:100) and anti-HMGB3 (cat:D160490, Sangon Biotech, China, 1:50). The reaction enhancer and the secondary antibody were added drop wise on the following day, and tissues were incubated for 1 h at room temperature. HMGB3 and CD34 expression were observed using 3,3′-diaminobenzidine, and samples were stained with hematoxylin and eosin. In brief, we measured the MVD, by dividing each xenograft into three layers and cutting three tissue slices from each layer. MVD was evaluated as previously described by Foote^{30 (link)}. We found areas with a high MVD under a low magnification (40×). Then, single microvessels were counted in three fields under a magnification of 200× or 400×.

Fresh tissues were immobilized with 4% formalin for 48 h at 37°C and subsequently washed with cool phosphate-buffered saline (PBS) three times for 5 min each time. Tissue samples were dehydrated using ethanol, embedded in paraffin, and then cut them into 4-μm-thick sections of 2 mm diameter to produce TMAs. The paraffin sections were dewaxed in an incubator at 65°C overnight, before being deparaffinized in xylene and rehydrated using a decreasing ethanol gradient. Sections were placed in ethylene diamine tetraacetic acid (cat.no. 0085; Beyotime) and heated in a microwave for 10 min for antigen repair. A 3% hydrogen peroxide was used to eliminate endogenous peroxidase at 37°C for 30 min. Sections were incubated with anti-HNRNPC (dilution 1:50, cat. no. ET1611-2; HUABIO), anti-ki67 (dilution 1:10000, cat. no. 27309; Proteintech), anti-CD34 (dilution 1:50, cat. no. ET1606-11; HUABIO), anti-Vimentin (dilution 1:100, cat. no. ET1610-39; HUABIO), and anti-E-Cadherin (dilution 1:50, cat. no. ET1607-75; HUABIO) antibodies at room temperature overnight, and then with secondary antibody (dilution: 1:1, cat.no. K5007; Dako) for 1 h at room temperature. Finally, sections were stained with an IHC kit (cat. no. K5007; Dako) according to the manufacturer’s protocol.