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[IF=2.447] Mei-Hua Jin. et al. Hispidin inhibits LPS‑induced nitric oxide production in BV‑2 microglial cells via ROS‑dependent MAPK signaling. Exp Ther Med. 2021 Sep;22(3):1-9  WB ;  Mouse.  

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Sample:
Lane 1: NIH/3T3 cell lysate, treated with Anisomycin
Lane 2: NIH/3T3 cell lysate, untreated
Primary: Anti-Phospho-JNK1/2/3(T183+T183+T221) (bsm-52462R) at 1:500 dilution
Secondary: Goat Anti-Rabbit IgG - HRP at 1:5000 dilution
Predicted band size: 42 kD
Observed band size: 54/46 kD

Paraformaldehyde-fixed, paraffin embedded (human Uterine Corpus 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 (Phospho-JNK123(T183+T183+T221)) Monoclonal Antibody, Unconjugated (bsm-52462R) at 1:200 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 (Phospho-JNK123(T183+T183+T221)) Monoclonal Antibody, Unconjugated (bsm-52462R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human prostate); 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 (Phospho-JNK123(T183+T183+T221)) Monoclonal Antibody, Unconjugated (bsm-52462R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat kidney); 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 (Phospho-JNK123(T183+T183+T221)) Monoclonal Antibody, Unconjugated (bsm-52462R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

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 ( Phospho-JNK1/2/3(T183+T183+T221)) monoclonal Antibody, Unconjugated (bsm-52462R) 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.

NIH/3T3 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 (Phospho-JNK1/2/3(T183+T183+T221)) monoclonal Antibody, Unconjugated (bsm-52462R) 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.

HUVEC 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 ( Phospho-JNK1/2/3(T183+T183+T221)) monoclonal Antibody, Unconjugated (bsm-52462R) 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.

Blank control:Hela.
Primary Antibody (green line): Rabbit Anti-Phospho-JNK1/2/3(T183+T183+T221) antibody (bsm-52462R)
Dilution: 1:100;
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 90% ice-cold methanol for 20 min at-20℃.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.