Blank control: K562. Primary Antibody (green line): Rabbit Anti-TLR4 antibody (orb11489), Dilution: 0.5 ug/Test, Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF488, Dilution: 0.5 ug/Test. Isotype control (orange line): Normal Rabbit IgG, Protocol, The cells were incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.
Blank control: Raw264.7. Primary Antibody (green line): Rabbit Anti-TLR4 antibody (orb11489), Dilution: 2 µg/10 6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-AF488, Dilution: 1 µg/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 0.1% PBST for 20 min at at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.
HL60 and MCF-7 cells were stained with rabbit polyclonal antibody against TLR4 with two dilutions (1:100 and 1:250). 2nd antibody without primary antibody was used as control included here.
Paraformaldehyde-fixed, paraffin embedded (Human esophageal cancer), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37C for 30 min, Antibody incubation with (TLR4) Polyclonal Antibody, Unconjugated (orb11489) at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Sample: Lane 1: Human MDA-MB-231 cell lysates, Lane 2: Human HUVEC cell lysates, Lane 3: Human HL-60 cell lysates, Primary: Anti-TLR4 (orb11489) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 90 kDa, Observed band size: 120 kDa.
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