Ab2435

For ELISA, flat-bottom Immune FEP-101 96-well plates (JET BIOFIL, Guangzhou, China) were coated overnight with 0.1 μg per well of the anti-TRAIL mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Plates were washed three times in PBS containing 0.2% Tween20 (PBST), blocked with PBST containing 2% BSA and then incubated in triplicate with diluted plasma or diluted tissue homogenate supernatant for 2 h at 37 °C. Plates were washed again and incubated with a 1/2000 dilution of a rabbit polyclonal antibody (ab2435; Abcam, Cambridge, MA, USA) to TRAIL for 2 h at 37 °C. The plates were then incubated with a 1/5000 dilution of a horse radish peroxidase-conjugated goat anti-rabbit secondary antibody (Proteintech Group, Inc., Chicago, IL, USA). After the final wash, plates were developed with 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB, QIAGEN, Hilden, Germany) for 20 min at room temperature and stopped after 10 min by adding 50 μl of 2M H₂SO_4. Analysis was performed using double wavelengths 450–630 nm with an EL × 800 Universal Microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

After the imaging procedures, the solitary xenograft tumors were fixed in 4% buffered formalin. Hematoxylin and eosin (H&E) staining was performed on serial sections of 4 μm intervals for histological examination. The surface and inner samples from these samples were frozen and sectioned to detect TRAIL and EGFP expression, as previously described [28 (link)]. The sections were blocked with normal goat serum for 30 minutes (Zli-9021, ZSGB-BIO, Beijing) and then incubated simultaneously with a 1:100-diluted polyclonal rabbit anti-TRAIL primary antibody (AB2435, Abcam, U.S.A.) and a 1:20-diluted polyclonal mouse anti-EGFP primary antibody (AB16278, Abcam, U.S.A.). Our detection of TRAIL-MSCs was based on incubation with a FITC-conjugated goat anti-rabbit secondary antibody (ZF0311, ZSGB-BIO, Beijing) and a rhodamine-conjugated goat anti-mouse secondary antibody (ZF0313, ZSGB-BIO, Beijing).