The slides were dewaxed in xylene, rehydrated, and then subjected to antigen retrieval in citrate buffer at 99°C for 5 min×3. For IF, the primary antibody anti-GM130 (Golgi matrix protein of 130kDa) (1:10, BD Pharmingen, USA) was incubated overnight at 4°C. Nuclear was stained using DAPI. For IHC, the primary antibodies, anti-phospho-JNK (Tyr183/Tyr185) or anti- JNK (1:50, Cell Signaling Technology Inc., MA, USA) were incubated overnight at 4°C; next procedures were performed with SP9001 kit and DAB Staining Kit (ZSGB-Bio, Beijing, China).
Anti phospho jnk tyr183 tyr185
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-JNK (Tyr183/Tyr185) is a laboratory reagent that detects the phosphorylated form of the c-Jun N-terminal kinase (JNK) protein. JNK is a member of the mitogen-activated protein kinase (MAPK) family and is activated by phosphorylation at Tyr183 and Tyr185.
Automatically generated - may contain errors
Lab products found in correlation
Sp9001 kit, by ZSGB-BIO (2 mentions)
Anti gm130, by BD (2 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Dab staining kit, by ZSGB-BIO (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti rabbit secondary antibody, by Cell Signaling Technology (2 mentions)
Anti gm130, by Cell Signaling Technology (2 mentions)
4 protocols using anti phospho jnk tyr183 tyr185
1
Immunofluorescence and Immunohistochemistry Protocols
2
Extraction and Analysis of Tooth Germ Tissues
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Before RNA extraction and protein extraction, E17.5 and P1 tooth germ were separated into mesenchymal and epithelial tissue; the separation process was performed as previously described [15 ].
Total RNA was isolated from E17.5, P1 separated tooth epithelial, and mesenchymal tissues using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s protocol. Next, cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, Dalian, China) and measured using a CFX96™ real-time PCR detection system (Bio-Rad, USA) as previously described [16 ]. Primer sequences were searched from PrimerBank (S1 Table ). Relative gene expression levels are presented as the mean and standard deviation from three independent experiments.
Total proteins of E17.5 and P1 tooth germ mesenchymal tissues were isolated, measured, transferred to a membrane, and incubated with anti-GAPDH, anti-phospho-JNK (Tyr183/Tyr185) (1:1000), anti-GM130 (1:500), and HRP-conjugated anti-rabbit secondary antibodies (1:1000, Cell Signaling Technology Inc., MA, USA). Finally, we determined the ratio of the target protein to the reference protein. The obtained ratios are presented as the mean and standard deviation from three independent experiments.
Total RNA was isolated from E17.5, P1 separated tooth epithelial, and mesenchymal tissues using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s protocol. Next, cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, Dalian, China) and measured using a CFX96™ real-time PCR detection system (Bio-Rad, USA) as previously described [16 ]. Primer sequences were searched from PrimerBank (
Total proteins of E17.5 and P1 tooth germ mesenchymal tissues were isolated, measured, transferred to a membrane, and incubated with anti-GAPDH, anti-phospho-JNK (Tyr183/Tyr185) (1:1000), anti-GM130 (1:500), and HRP-conjugated anti-rabbit secondary antibodies (1:1000, Cell Signaling Technology Inc., MA, USA). Finally, we determined the ratio of the target protein to the reference protein. The obtained ratios are presented as the mean and standard deviation from three independent experiments.
3
Gene and Protein Expression Analysis in Developing Tooth Germ
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Before RNA extraction and protein extraction, E17.5 and P1 tooth germ should be separated into mesenchymal and epithelial tissue, and the separation methods were performed as previously described [16] .
Total RNAs were isolated from E17.5 and P1 separated tooth epithelial and mesenchymal tissues using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) according to manufacturer's protocol. After cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, Dalian, China) and measured using a CFX96™ real-time PCR detection system (Bio-Rad, USA) as described previously [17] . Primer sequences are searched from PrimerBank (S1 Table ). Relative gene expression levels are presented as the mean and standard deviation from three independent experiments. Total proteins of E17.5 and P1 tooth germ mesenchymal tissues were isolated, and protein concentration was measured, protein transfer and incubate following regular methods. Anti-GAPDH, anti-phospho-JNK (Tyr183/Tyr185) (1:1000), anti-GM130 (1:500), and HRP-conjugated anti-rabbit secondary antibodies (1:1000, Cell Signaling Technology Inc., MA, USA) were used. Finally, we determined the ratio of the target protein to the reference protein. The obtained ratios are presented as the mean and standard deviation from three independent experiments.
Total RNAs were isolated from E17.5 and P1 separated tooth epithelial and mesenchymal tissues using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) according to manufacturer's protocol. After cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, Dalian, China) and measured using a CFX96™ real-time PCR detection system (Bio-Rad, USA) as described previously [17] . Primer sequences are searched from PrimerBank (S1 Table ). Relative gene expression levels are presented as the mean and standard deviation from three independent experiments. Total proteins of E17.5 and P1 tooth germ mesenchymal tissues were isolated, and protein concentration was measured, protein transfer and incubate following regular methods. Anti-GAPDH, anti-phospho-JNK (Tyr183/Tyr185) (1:1000), anti-GM130 (1:500), and HRP-conjugated anti-rabbit secondary antibodies (1:1000, Cell Signaling Technology Inc., MA, USA) were used. Finally, we determined the ratio of the target protein to the reference protein. The obtained ratios are presented as the mean and standard deviation from three independent experiments.
4
Immunostaining for Golgi and Kinase
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The slides were dewaxed in xylene, rehydrated and then subjected to antigen retrieval in citrate buffer at 99 ℃ for 5 min×3. For IF, the primary antibody anti-GM130 (Golgi matrix protein 130) (1:10, BD Pharmingen, USA) was incubated overnight at 4°C. Nuclear was stained using DAPI. For IHC, the primary antibodies, anti-phospho-JNK (Tyr183/Tyr185) or anti-JNK (1:50, Cell Signaling Technology Inc., MA, USA)
were incubated overnight at 4°C, and the next procedures were performed with SP9001 kit and DAB Staining Kit (ZSGB-Bio, Beijing, China).
were incubated overnight at 4°C, and the next procedures were performed with SP9001 kit and DAB Staining Kit (ZSGB-Bio, Beijing, China).
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