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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).

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Immunohistochemical analysis of paraffin-embedded Kelly xenograft using Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb #9579 (left) or Rabbit (DA1E) mAb IgG XP® SignalStain® Isotype Control #12960 (right).

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer.

    NOTE: Additional 10X TBST will be required for washes.

    1. 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
    2. 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.
  6. Antibody Diluent: SignalStain® Antibody Diluent #8112
  7. Antigen Unmasking: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O72H2O) to 1 L dH2O. Adjust pH to 6.0.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. 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.
  10. Detection System: SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (#8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059), which includes SignalStain® DAB Diluent (#11724) and SignalStain® DAB Chromogen Concentrate (#11725).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections twice in dH2O for 5 min each.

C. Antigen Unmasking

  1. 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.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O twice for 5 min each.
  4. Wash section in wash buffer for 5 min.
  5. Block each section with 100–200 μl blocking solution for 1 hr at room temperature.
  6. 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.
    1. Rabbit (DA1E) mAb XP® SignalStain® Isotype Control (#3900) is used as a negative control. Dilute at 1:500 in SignalStain® Antibody Diluent (#8112) and apply 100–200 μl to each section.
  7. Equilibrate SignalStain® Boost Detection Reagent (#8114) to room temperature.
  8. Remove antibody solution and wash sections in wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (#8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 30 μl SignalStain® DAB Chromogen Concentrate (#11725) to 1 ml SignalStain® DAB Diluent (#11724) and mix well before use.
  12. Apply 100–400 μl SignalStain® DAB to each section and monitor closely. 1–10 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount coverslips.

posted July 2013

protocol id: 64

Product Includes Quantity Isotype
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  

Order Details

Custom Ordering Details: The control slides that accompany this kit are cut freshly upon ordering. Please allow three to five days for your order to be processed and shipped.

Product Description

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.


Specificity / Sensitivity

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

Source / Purification

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).


1.  Franke, T.F. et al. (1997) Cell 88, 435-7.

2.  Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.

3.  Franke, T.F. et al. (1995) Cell 81, 727-36.

4.  Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.

5.  Sarbassov, D.D. et al. (2005) Science 307, 1098-101.

6.  Jacinto, E. et al. (2006) Cell 127, 125-37.

7.  Cardone, M.H. et al. (1998) Science 282, 1318-21.

8.  Brunet, A. et al. (1999) Cell 96, 857-68.

9.  Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.

10.  Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.

11.  Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.

12.  Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.

13.  Cross, D.A. et al. (1995) Nature 378, 785-9.

14.  Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.

15.  Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.

16.  Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.

17.  Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.

18.  Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.

19.  Manning, B.D. et al. (2002) Mol Cell 10, 151-62.


Entrez-Gene Id 207, 208, 10000
Swiss-Prot Acc. P31749, P31751, Q9Y243

Protein Specific References

Germack R and Dickenson JM (2000) Br J Pharmacol 130, 867–74

Wick MJ et al. (2000) J Biol Chem 275, 40400–6

Rane MJ et al. (2001) J Biol Chem 276, 3517–23

Guizzetti M and Costa LG (2001) Neuroreport 12, 1639–42

Brognard J et al. (2001) Cancer Res 61, 3986–97

Maira SM et al. (2001) Science 294, 374–80

Schönherr E et al. (2001) J Biol Chem 276, 40687–92

Hill MM et al. (2001) J Biol Chem 276, 25643–6

Dhawan P et al. (2002) Cancer Res 62, 7335–42

Conus NM et al. (2002) J Biol Chem 277, 38021–8

Sano H et al. (2002) J Biol Chem 277, 19439–47

Egawa K et al. (2002) J Biol Chem 277, 38863–9

Kisseleva MV et al. (2002) J Biol Chem 277, 6266–72

Barry FA and Gibbins JM (2002) J Biol Chem 277, 12874–8

Ikonomov OC et al. (2002) Endocrinology 143, 4742–54

Rani MR et al. (2002) J Biol Chem 277, 38456–61

Ho R et al. (2002) Cancer Res 62, 6462–6

Wan X and Helman LJ (2003) Oncogene 22, 8205–11

Fukuda T et al. (2003) J Biol Chem 278, 51324–33

Kim HH et al. (2003) FASEB J 17, 2163–5

Min YH et al. (2004) Cancer Res 64, 5225–31

Tazzari PL et al. (2004) Br J Haematol 126, 675–81

Matsuzaki H et al. (2004) Biochemistry 43, 4284–93

Wolfrum S et al. (2004) Arterioscler Thromb Vasc Biol 24, 1842–7

Kaneko Y et al. (2004) J Cell Sci 117, 407–15

Esfandiarei M et al. (2004) J Virol 78, 4289–98

Baudhuin LM et al. (2004) FASEB J 18, 341–3

Dietze EC et al. (2004) Oncogene 23, 3851–62

Wu T et al. (2004) Mol Cancer Ther 3, 299–307

Honjo S et al. (2005) DNA Cell Biol 24, 141–7

Karlsson HK et al. (2005) Diabetes 54, 1459–67

Viniegra JG et al. (2005) J Biol Chem 280, 4029–36

Le XF et al. (2005) J Biol Chem 280, 2092–104

Smith E and Frenkel B (2005) J Biol Chem 280, 2388–94

Edwards LA et al. (2005) Oncogene 24, 3596–605

Karlsson HK et al. (2005) Diabetes 54, 1692–7

Kippenberger S et al. (2005) J Biol Chem 280, 3060–7

Jung HS et al. (2005) Mol Endocrinol 19, 2748–59

Khundmiri SJ et al. (2006) Am J Physiol Cell Physiol 291, C1247–57

Hers I and (2007) Blood 110, 4243–52

Ananthanarayanan B et al. (2007) J Biol Chem 282, 36634–41

Zunder ER et al. (2008) Cancer Cell 14, 180–92

Grenegård M et al. (2008) J Biol Chem 283, 18493–504

Abubaker J et al. (2009) Mol Cancer 8, 51

Chen PL and Easton AS (2011) Curr Neurovasc Res 8, 14–24

Van Aller GS et al. (2011) Biochem Biophys Res Commun 406, 194–9

Uesugi A et al. (2011) Cancer Res 71, 5765–78

Ou YH et al. (2011) Mol Cell 41, 458–70

Wang S et al. (2012) PLoS One 7, e37427

Glidden EJ et al. (2012) J Biol Chem 287, 581–8

Shih MC et al. (2012) Oncogene 31, 2389–400

Misra UK and Pizzo SV (2012) J Cell Biochem 113, 1488–500

Johnson AL et al. (2001) Biol Reprod 64, 1566–74

Zhang M and Riedel H (2009) J Cell Biochem 107, 65–75


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.