Anti PD-L1  

Figure 1 Western Blot Validation of PD-L1 in HeLa Cells
Loading: 15 μg of lysates per lane.Antibodies: 4059 (A: 1 μg/mL, B: 2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Human and Mouse cell lines
Loading: 15 μg of lysates per lane.Antibodies: 4059 (2 μg/mL), RF16035 (2 μg/mL), and beta-actin (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit and or anti-mouse IgG HRP conjugate at 1:10000 and 1:5000 dilution, respectively.

Figure 3 Validation with PD-L1 siRNA Knockdown in HeLa Cells
HeLa cells were transfected with control siRNAs (lane 1) or PD-L1 siRNAs (lane 2) Loading: 10 μg of HeLa whole cell lysates per lane.Antibodies: 4059 (2 μg/mL) and GAPDH (3783, 0.02 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-mouse IgG HRP conjugate at 1:5000 dilution.

Figure 4 Validation with PD-L1 overexpression in 293 cells
Loading: Lysates/proteins at 15 μg per lane.Lane 1: non-transfected 293 cellsLane 2: PD-L1 overexpressed 293 cellsAntibodies: 4059 (1 μg/mL). 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Figure 5 Immunohistochemistry Validation of PD-L1 in Human Tonsil Cells
Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-PD-L1 antibody (4059) at 5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4℃. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

Figure 6 Immunofluorescence Validation of PD-L1 in Human Heart
Immunofluorescent analysis of 4% paraformaldehyde-fixed human heart tissue labeling PD-L1 with 4059 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing both membrane and cytoplasmic staining on human heart tissue.

Figure 7 Flow Cytometry Validation of PD-L1
Overlay histogram showing A-20 cells stained with 4059 (red line, 1μg/1x10⁶ cells). 1 h incubation at 4℃ in 2% FBS/PBS. Followed by secondary antibody 488 goat anti-rabbit IgG (H+L) at 1/500 dilution for 1 h 4℃. Isotype control antibody (Green line) was mouse IgG1 (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >10,000 events was performed.

Figure 8 Immunohistochemistry Validation of PD-L1 in Rat Heart
Immunohistochemical analysis of paraffin-embedded rat heart tissue using anti-PD-L1 antibody (4059) at 5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4℃. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

Figure 9 Immunohistochemistry Validation of PD-L1 in Human Heart
Immunohistochemical analysis of paraffin-embedded human heart tissue using anti-PD-L1 antibody (4059) at 2.5 μｋg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4℃. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

Figure 10 Immunofluorescence Validation of PD-L1 in Rat Heart
Immunofluorescence analysis of 4% paraformaldehyde-fixed rat heart tissue labeling PD-L1 with 4059 at 20 μg/ml, followed by goat anti-rabbit IgG secondary antibody at 1/250 dilution (red).