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Phospho-Akt (Ser473) Antibody Staining Protocol for Immunohistochemistry
Description: Akt, also referred to as PKB or Rac, plays a critical role in controlling the balance between survival and apoptosis. This protein kinase is activated by insulin and various growth and survival factors, and functions in a wortmannin-sensitive pathway involving PI3 kinase. Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1, and by phosphorylation within the carboxyterminus at Ser473. Akt promotes cell survival by inhibiting apoptosis through its ability to phosphorylate and inactivate several targets, including Bad, Forkhead transcription factors and caspase-9. PTEN phosphatase is a major negative regulator of the PI3kinase/Akt signaling pathway.
Primary Antibody
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Name: Phospho-Akt (Ser 473) (736E11) Antibody |
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Clone: Rabbit anti-Human |
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Supplier: Cell Signaling Technology |
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Catalog Number: 3787 |
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Dilution: 1:100 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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Incubation Time/Temp: 60 min/room temperature |
Antigen Retrieval
| Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) |
| Heat/Cool Temperature: 95-100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cytoplasmic/Nuclear |
| Images: Search image |
Additional Information:
| Species Reactivity: Human, mouse, rat |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Breast carcinoma, lung carcinoma, prostate carcinoma, LNCaP cells |
| Negative Control: Omit primary antibody, isotype control, absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
References:
1. Hall PA, et al (1990) Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol 162:285-294.
2. Yu CCW, et al (1991) Haemangiopericytomas: the prognostic value of immunohistochemical staining with a monoclonal antibody to proliferating cell nuclear antigen (PCNA). Histopathol 19:29-33.