Anti c5b 9 antibody

Quantification of C5b-9 using cell-ELISA technique was performed as previously described [24 (link)]. Briefly, cell-ELISA was performed in 96-well culture plates (BD Biosciences, Franklin Lakes, NJ, USA). HUVECs were seeded at 10,000 cells/well and incubated overnight at 37 °C in 5% CO2. Cells were then cultured in media containing 10% NS at 37 °C in 5% CO₂ for 1 h to activate the complement system. Cells were fixed with 3% paraformaldehyde. A rabbit polyclonal anti-C5b-9 antibody (Abcam, Cambridge, MA, USA) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) were used as primary and secondary antibody, respectively. 3,3′,5,5′-tetramethylbenzidine (TMB, KPL, Gaithersburg, MD, USA) was added as a substrate, and the absorbance at 450 nm was measured using a microplate reader (Molecular Devices, Silicon Valley, CA, USA).

To examine the formation of the membrane attack complex in cells treated with EVs under NHS‐induced complement activation, PLC/PRF/5 cells were seeded on coverslips and incubated with EVs for 72 h followed by NHS stimulation. The EV‐treated cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton‐X‐100 in PBS and blocked with 3% bovine serum albumin in PBS at room temperature. The cells were then subjected to incubation with 1:500 anti‐C5b‐9 antibody (Abcam) and subsequent incubation with 1:200 Goat anti‐Rabbit IgG (H+L) Cross‐Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher) and DAPI (4′,6‐diamidino‐2‐phenylindole, dihydrochloride) (Thermo Fisher). The processed coverslips were mounted in Vectashield anti‐fade mounting medium (Vector Laboratories). Images were captured by ZEISS LSM 900 confocal microscope (Carl Zeiss).

Human paraffin embedded tissue array slide for bone and cartilage cancer tissue (BO241) was purchased from US Biomax, Inc (Derwood, MD). Tissue sections were deparaffinized in xylene followed by a graded series of alcohol washes prior to staining. Subsequently, all sections were treated with 3% H₂O₂ for 10 minutes to block endogenous peroxidase activity, and then slides were blocked with normal goat serum (Vector laboratories, Burlingame, CA) for 1 h at room temperature (RT). After blocking, slides were incubated with the anti-C5b-9 antibody (1:100, Abcam Inc., Cambridge, MA) overnight at 4 °C. Then sections incubated with biotinylated goat anti-rabbit secondary antibodies (1:100, Vector Laboratories, Burlingame, CA) for 1 h at RT, followed by 30 min incubation with Vectastain avidin-biotin complex reagents (Vectastain-Elite kit, Vector Laboratories, Burlingame, CA). Then color was developed with 3,3′-diaminobenzidine(DAB) and counterstained with Mayer’s hematoxylin. Finally, Slides were observed under an Eclipse E400 microscope (Nikon Instruments Inc., USA) and images were captured with a Nikon Digital Sight DS-U2 camera.