VALIDATION IMAGES
Human U937 Cells were fixed with 2% PFA (10min at room temperature). The cells were then incubated in 1X PBS containing 0.5% BSA + 10% goat serum to block non-specific protein-protein interactions for 15 min at room temperature. Staining was performed with CD86 Polyclonal Antibody, Unconjugated (bs-1035R) at 1:100 for 30 min on ice. A Goat Anti-rabbit IgG-PE secondary antibody was used for a 30 min incubation on ice. Primary antibody staining (green) is compared to unstained cells (dark blue), secondary antibody only (light blue) and isotype control (bs-0295P; orange).
Mouse Brain cells probed with CD86 Polyclonal Antibody, PE Conjugated (bs-1035R-PE; green) at 1:50 compared to isotype control (bs-0295P-PE; orange).
L1 rat brain lysates L2 rat heart lysates probed with Anti CD86/B7-2 Polyclonal Antibody, Unconjugated (bs-1035R) at 1:200 overnight at 4˚C. Followed by conjugation to secondary antibody (bs-0295G-HRP) at 1:3000 for 90 min at RT. Predicted band 34kD. Observed band size:34kD.
Raji cell lysates probed with CD86 Polyclonal Antibody, Unconjugated (bs-1035R) at 1:300 overnight at 4˚C. Followed by conjugation to secondary antibody (bs-0295G-HRP) at 1:500 for 90 min at 37˚C.
Mouse BV-2 Cells were fixed with 4% PFA (10min at room temperature) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature. The cells were then incubated in 1X PBS / 2% BSA / 10% goat serum to block non-specific protein-protein interactions for 20 min at 37°C. The cells were stained with CD86 Polyclonal Antibody Unconjugated (bs-1035R; green) at 1:100 for 90 min at 37°C. A FITC-conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-FITC) was incubated at 37°C for 90 minutes. DAPI (5ug/ml; blue) was used to stain the cell nuclei.
Lane 1: Human HepG2 cell lysates; Lane 2: Human U937 cell lysates; Lane 3: Rat Spleen lysates; Lane 4: Mouse Spleen lysates; Lane 5: Human HL-60 cell lysates probed with CD86 Polyclonal Antibody, Unconjugated (bs-1035R) at 1:1000 dilution and 4˚C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.
The Daudi(H) cells were incubated in 5%BSA to block non-specific protein-protein interactions (30 min at r.t.). Primary Antibody (green): Rabbit Anti-CD86 antibody (bs-1035R): 1 μg/10^6 cells; Secondary Antibody (white blue): Goat anti-Rabbit IgG-FITC (bs-60295G-FITC): 1 μg/test. Isotype Control (orange): Rabbit IgG (bs-0295P). Blank control (black): PBS. Acquisition of 20,000 events was performed.