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 (bs-1021R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 90 kDa
Observed band size: 120 kDa

Paraformaldehyde-fixed, paraffin embedded (Human esophageal cancer); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (TLR4) Polyclonal Antibody, Unconjugated (bs-1021R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Tissue/cell: rat heart tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-TLR4/CD284 Polyclonal Antibody, Unconjugated(bs-1021R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Image submitted by One World Lab validation program. 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.

Blank control:K562.
Primary Antibody (green line): Rabbit Anti-TLR4 antibody (bs-1021R)
Dilution: 0.5ug/Test;
Secondary Antibody (white blue line) : Goat anti-rabbit IgG-AF488
Dilution: 0.5ug/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 20,000 events was performed.

Blank control: Raw264.7.
Primary Antibody (green line): Rabbit Anti-TLR4 antibody (bs-1021R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF488
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min 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 20,000 events was performed.