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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human transitional epithelial carcinoma of the bladder, using Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific) (#2976) in the presence of control peptide (left) or Phospho-mTOR (Ser2448) Blocking Peptide (right).

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
    2. Antibody Diluent TBST/5% Normal Goat Serum: to 5 mL 1X TBST, add 250 µl Normal Goat Serum #5425).
  6. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  7. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  8. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  9. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  10. Substrate: Vector® NovaRED™ (Vector Laboratories).
  11. Hematoxylin: Hematoxylin (#14166).
  12. Mounting Medium: SignalStain® Mounting Medium (#14177).

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 two times in dH2O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). 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 two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in recommended antibody diluent to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #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. Prepare Vector® NovaRED™ per manufacturer's recommendations.
  12. Apply 100-400 µl substrate 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 with hematoxylin (#14166).
  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 sections with coverslips and mounting medium (#14177).

posted February 2010

revised March 2016

Protocol Id: 305

Product Description

This peptide is used to specifically block Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific) #2976 and #2971 Phospho-mTOR (Ser2448) Antibody reactivity.


Quality Control

The quality of the peptide was evaluated by reversed-phase HPLC and by mass spectrometry. The peptide blocks Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific) (#2976) signal in immunohistochemistry and #2971 Phospho-mTOR (Ser2448) Antibody in western blotting.

Product Usage Information

Use as a blocking reagent to evaluate the specificity of antibody reactivity in immunohistochemistry protocols. For immunohistochemistry, add twice the volume of peptide as volume of antibody used in 100 ul total volume. Incubate for a minimum of 30 minutes prior to adding the entire volume to the slide. For Western blotting, add an equal volume of peptide as volume of antibody used in 10 ml total volume, and incubate at room temperature for 30 minutes before allowing to react with the blot. Recommended antibody dilutions can be found on the product data sheet.


Storage: Supplied in 20 mM potassium phosphate (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 1 mg/ml BSA, 5% glycerol and 1% DMSO. Store at –20°C.

The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (1-3) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (4,5). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (6). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (7,8). mTOR plays a key role in cell growth and homeostasis and may be abnormally regulated in tumors. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy (9).


No product specific background information is currently available for this product.


1.  Sabers, C.J. et al. (1995) J Biol Chem 270, 815-22.

2.  Brown, E.J. et al. (1994) Nature 369, 756-8.

3.  Sabatini, D.M. et al. (1994) Cell 78, 35-43.

4.  Gingras, A.C. et al. (2001) Genes Dev 15, 807-26.

5.  Dennis, P.B. et al. (2001) Science 294, 1102-5.

6.  Fang, Y. et al. (2001) Science 294, 1942-5.

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

8.  Peterson, R.T. et al. (2000) J Biol Chem 275, 7416-23.

9.  Huang, S. and Houghton, P.J. (2003) Curr Opin Pharmacol 3, 371-7.


Entrez-Gene Id 2475
Swiss-Prot Acc. P42345


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

1230
Phospho-mTOR (Ser2448) Blocking Peptide