Sample:
Lane 1: Human THP-1 cell Lysates
Lane 2: Human K562 cell Lysates
Lane 3: Human U937 cell Lysates
Lane 4: Human U87MG cell Lysates
Lane 5: Human HUVEC cell Lysates
Primary: Anti-TNFR2 (bsm-52938R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 46kDa
Observed band size: 70 kDa

Sample:
Lane 1: MCF-7 cell lysate
Lane 2: Jurkat cell lysate
Lane 3: Hela cell lysate
Primary: Anti-TNFR2 (bsm-52938R) at 1:500 dilution
Secondary: Goat Anti-Rabbit IgG - HRP at 1:5000 dilution
Predicted band size: 46 kD
Observed band size: 60 kD

Paraformaldehyde-fixed, paraffin embedded (mouse kidney tissue); 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 (TNFR2) Monoclonal Antibody, Unconjugated (bsm-52938R) at 1:50 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human uterus tissue); 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 (TNFR2) Monoclonal Antibody, Unconjugated (bsm-52938R) at 1:50 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human kidney tissue); 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 (TNFR2) Monoclonal Antibody, Unconjugated (bsm-52938R) at 1:50 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Blank control:HL-60.
Primary Antibody (green line): Rabbit Anti-TNF Receptor II antibody (bsm-52938R)
Dilution: 1:50;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF488
Dilution: 1:1000.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 0.1% PBST for 20 min 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.

SW480 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (TNF Receptor II) monoclonal Antibody, Unconjugated (bsm-52938R) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (TNF Receptor II) monoclonal Antibody, Unconjugated (bsm-52938R) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

MCF-7 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (TNF Receptor II) monoclonal Antibody, Unconjugated (bsm-52938R) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.