Sample: Lane 1: Mouse Brain Lysates Lane 2: Mouse Pancrease Lysates Lane 3: Mouse Liver Lysates Lane 4: Mouse NIH/3T3 cell Lysates Lane 5: Rat Brain Lysates Lane 6: Rat Pancrease Lysates Lane 7: Human HUVEC cell Lysates Lane 8: Human U-87 MG cell Lysates Lane 9: Human SH-SY5Y cell Lysates Primary: Anti-DUSP6 (bsm-52270R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 42kDa Observed band size: 42kDa

Sample: Lane 1: mouse pancreas tissue lysates Primary: Anti-DUSP6 (bsm-52270R) at 1:500 dilution Secondary: Goat Anti-Rabbit IgG - HRP at 1:5000 dilution Predicted band size: 42 kD Observed band size: 55 kD

Paraformaldehyde-fixed, paraffin embedded (mouse brain); 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 (DUSP6) Monoclonal Antibody, Unconjugated (bsm-52270R) 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 gastric carcinoma); 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 (DUSP6) Monoclonal Antibody, Unconjugated (bsm-52270R) at 1:50 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse pancreas); 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 (DUSP6) Monoclonal Antibody, Unconjugated (bsm-52270R) 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 pancreas); 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 (DUSP6) Monoclonal Antibody, Unconjugated (bsm-52270R) at 1:50 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

NIH/3T3 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with ( DUSP6 (9C3)) Monoclonal Antibody, Unconjugated (bsm-52270R) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) 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) at 37°C for 20 min; Antibody incubation with (DUSP6 (9C3) )Monoclonal Antibody, Unconjugated (bsm-52270R) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.

PC12 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (DUSP6 (9C3) )Monoclonal Antibody, Unconjugated (bsm-52270R) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.

Blank control:NIH/3T3. Primary Antibody (green line): Rabbit Anti-DUSP6 antibody (bsm-52270R) 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.