VALIDATION IMAGES
Formalin-fixed and paraffin embedded: human lung carcinoma labeled with Anti-JNK1/3 Polyclonal Antibody (bs-0501R), Unconjugated at 1:200 followed by conjugation to the secondary antibody and DAB staining
Image kindly provided by Dr. Magdalena Krol. Control tumor cells, tumor cells grown in macrophage-conditioned medium, tumor cells sorted from co-culture with macrophages, and macrophages from monocultures and sorted from co-culture with tumor cells were analyzed. Total protein concentrations in lysates were determined using a Bio-Rad protein assay. Proteins (50 mg) were resolved using SDS-PAGE and transferred onto PVDF membranes. The membranes were then blocked with 5% non-fat dry milk in TBS buffer containing 0.5% Tween 20. The membranes were then incubated overnight with the primary Rabbit Anti-JNK1 + 3 Polyclonal Antibody at 1:100 dilution. Subsequently, the membranes were washed three times in TBS containing 0.5% Tween 20 and incubated for 1 h at room temperature with secondary antibodies conjugated with the appropriate infrared (IR) fluorophore IRDyeH 800 CW or IRDyeH 680 RD at a dilution of 1:5000.
Paraformaldehyde-fixed, paraffin embedded human lung carcinoma; Antigen retrieval by boiling in sodium citrate buffer (pH6) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (normal goat serum) at 37°C for 20min; Antibody incubation with \tJNK1 + 3 Polyclonal Antibody, Unconjugated (bs-0501R) at 1:500 overnight at 4°C, followed by a conjugated secondary and DAB staining.
Paraformaldehyde-fixed, paraffin embedded Rat uterus; 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 JNK1+3 Polyclonal Antibody, Unconjugated (bs-0501R) at 1:200 overnight at 4°C, DAB staining.
Paraformaldehyde-fixed, paraffin embedded Human stomach 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 JNK1 + 3 Polyclonal Antibody, Unconjugated (bs-0501R) at 1:200 overnight at 4°C, DAB staining.
Lane 1: Human Hela cell lysates; Lane 2: Human Jurkat cell lysates; Lane 3: Human K562 cell lysates; Lane 4: Mouse NIH/3T3 cell lysates; Lane 5: Mouse RAW264.7 cell lysates; Lane 6: Human A431 cell lysates; Lane 7: Mouse Uterus lysates; Lane 8: Rat Uterus lysates probed with JNK1 + 3 Polyclonal Antibody, Unconjugated (bs-0501R) at 1:1000 dilution and 4˚C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.
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 (JNK1 + JNK3) polyclonal Antibody, Unconjugated (bs-0501R) 1:100, 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.