Upstream / Downstream
Explore pathways related to this product.
CST Antibody Performance Guarantee
Find answers on our FAQs page.
PTM information and tools available.
Survival Marker: SignalStain® Phospho-Akt (Ser473) IHC Detection Kit #8100
This Product is Unavailable
Immunohistochemical analysis of paraffin-embedded E14 mouse embryo using Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb #9579 (left) or Rabbit (DA1E) mAb IgG XP® SignalStain® Isotype Control #12960 (right).Learn more about how we got this image
Gallery: Survival Marker: SignalStain® Phospho-Akt (Ser473) IHC Detection Kit #8100
A. Solutions and Reagents
- Ethanol, anhydrous denatured, histological grade (100% and 95%).
- Deionized water (dH2O).
- Hematoxylin (optional).
NOTE: Additional 10X TBST will be required for washes.
- Tris Buffered Saline with Tween® 20 (TBST-10X) #9997: To prepare wash buffer add 100 μl of Tris Buffered Saline with Tween® 20 (TBST-10X) #9997 to 900 ml of dH2O. OR
- 10X Tris Buffered Saline (TBS): To prepare 1 L, add 24.2 g Trizma® base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1 L dH2O. Adjust pH to 7.6 with concentrated HCl. 1X TBS/0.1% Tween® 20 (1X TBST): To prepare 1 L, add 100 ml 10X TBS to 900 ml dH2O. Add 1 ml Tween® 20 and mix.
- Antibody Diluent: SignalStain® Antibody Diluent #8112
- Antigen Unmasking: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
- 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: 1X TBST/5% normal goat serum: Add 100 μl 10X TBST (#9997) and 50 μl normal goat serum (#5425) to 850 μl dH2O.
- Detection System: SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (#8114).
- Substrate: SignalStain® DAB Substrate Kit (#8059), which includes SignalStain® DAB Diluent (#11724) and SignalStain® DAB Chromogen Concentrate (#11725).
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 min each.
- Incubate sections in two washes of 100% ethanol for 10 min each.
- Incubate sections in two washes of 95% ethanol for 10 min each.
- Wash sections twice in dH2O for 5 min each.
C. Antigen Unmasking
- Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0, then maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
- Wash sections in dH2O three times for 5 min each.
- Incubate sections in 3% hydrogen peroxide for 10 min.
- Wash sections in dH2O twice for 5 min each.
- Wash section in wash buffer for 5 min.
- Block each section with 100–200 μl blocking solution for 1 hr at room temperature.
- Remove blocking solution and add 100–200 μl primary antibody diluted at 1:500 in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
- Equilibrate SignalStain® Boost Detection Reagent (#8114) to room temperature.
- Remove antibody solution and wash sections in wash buffer three times for 5 min each.
- Cover section with 1–3 drops SignalStain® Boost Detection Reagent (#8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
- Wash sections three times with wash buffer for 5 min each.
- Add 30 μl SignalStain® DAB Chromogen Concentrate (#11725) to 1 ml SignalStain® DAB Diluent (#11724) and mix well before use.
- Apply 100–400 μl SignalStain® DAB to each section and monitor closely. 1–10 minutes generally provides an acceptable staining intensity.
- Immerse slides in dH2O.
- If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
- Wash sections in dH2O two times for 5 min each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 sec each.
- Repeat in 100% ethanol, incubating sections two times for 10 sec each.
- Repeat in xylene, incubating sections two times for 10 sec each.
- Mount coverslips.
posted July 2013
protocol id: 64
|Peroxidase Quench||1 x 19.5 ml|
|Blocking Solution||1 x 15 ml|
|Prediluted P-Akt (S473) Ab||1 x 15 ml|
|Prediluted Negative Control||1 x 15 ml|
|Biotinylated Secondary Ab||1 x 15 ml|
|NovaRED Substrate 1 (TM)||1 x 0.6 ml|
|NovaRED Substrate 2 (TM)||1 x 0.6 ml|
|NovaRED Substrate 3 (TM)||1 x 0.6 ml|
|NovaRED Substrate 4 (TM)||1 x 0.8 ml|
|Reagent A||1 x 0.85 ml|
|Reagent B||1 x 0.85 ml|
|Mixing Bottle||1 x|
|Phospho-Akt (Ser473) Blocking Peptide 1140||x 100 µg|
CST's Survival Marker: Signal Stain® Phospho-Akt (Ser473) IHC Detection Kit is a "ready to use" system designed to detect the activation of Akt in human tissue and cell preparations using immunohistochemistry. The kit utilizes the ABC immunoperoxidase method to detect endogenous levels of phosphorylated Akt protein. Prediluted Phospho-Akt (Ser473) Antibody is bound by a biotinylated secondary antibody. Avidin DH and biotinylated horseradish peroxidase are complexed by mixing defined amounts prior to use, and the mixture subsequently binds the secondary antibody. The macromolecular complex is localized by incubation with NovaRED™ enzyme substrate.
The prediluted primary antibody, along with the ABC system, allows the user to consistently examine phosphorylated-Akt localization and offers the highest sensitivity with the lowest background.
Survival Marker: Signal Stain® Phospho-Akt (Ser473) IHC Detection Kit detects Akt1 only when phosphorylated at serine 473, and Akt2 and Akt3 only when phosphorylated at equivalent sites. The antibody does not detect Akt phosphorylated at other sites or related kinases such as PKC or p70 S6 kinase. This kit was developed for and is recommended for immunohistochemistry only.Species predicted to react based on 100% sequence homology: Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser473 of mouse Akt. Antibodies are purified by protein A and peptide affinity chromatography.
Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).
Product Specific References
Protein Specific References
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. SignalStain® is a trademark of Cell Signaling Technology, Inc. NovaRED™ is a trademark of Vector Laboratories.