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PhosphoSitePlus® Resource

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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 289 Rabbit IgG
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Immunohistochemistry (Frozen)

Immunohistochemical analysis of paraffin-embedded human transitional epithelial carcinoma of the bladder using Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific) in the presence of control peptide (left) or Phospho-mTOR (Ser 2448) Blocking Peptide #1230 (right).

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Immunohistochemistry (Frozen)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Frozen)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma untreated (left) or lambda phosphatase-treated (right), using Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Frozen)

Immunohistochemical analysis of frozen H1650 xenograft, showing cytoplasmic localization, using Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC specific).

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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

Immunohistochemistry (Frozen)

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. Fixative: Acetone.
  5. Wash Buffer: 1X Tris Buffered Saline (TBS).

    To prepare 1L 1X TBS add 100 ml 10X Tris Buffered Saline (#12498) to 900 ml dH2O, mix.

  6. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  7. Blocking Solution: 1X TBS/0.3% Triton X-100/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. 1X TBS/0.3% Triton X-100/5% Normal Goat Serum: add 500 µl Normal Goat Serum (#5425) and 30 µl Triton X-100 to 9.5 ml 1X TBS.
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  8. Antibody Diluent 1X TBS/0.3% Triton™ X-100/5% Normal Goat Serum: add 500 µl Normal Goat Serum (#5425) and 30 µl Triton™ X-100 to 9.5 ml 1X TBS.
  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. Sectioning

  1. For tissue stored at -80°C: Remove from freezer and equilibrate at -20°C for approximately 15 min before attempting to section. This may prevent cracking of the block when sectioning.
  2. Section tissue at a range of 6–8 µm and place on positively charged slides.
  3. Allow sections to air dry on bench for a few min before fixing (this helps sections adhere to slides).

C. Fixation

  1. Fix sections in cold acetone for 10 min at -20°C. Air dry. Proceed with staining procedure immediately (Section D).

D. Staining

  1. Wash sections in wash buffer two times for 5 min.
  2. Incubate for 10 min at room temperature in 3% Hydrogen Peroxide.
  3. Wash sections in wash buffer two times for 5 min.
  4. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  5. Remove blocking solution and add 100–400 µl primary antibody diluted in recommended antibody diluent to each section.
  6. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #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 (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™ (Vector Laboratories) per manufacturer's instructions.
  12. Apply 100-400 µl substrate to each section and monitor staining 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: 326

Product Usage Information

Application Dilutions
Immunohistochemistry (Paraffin) 1:100
Immunohistochemistry (Frozen) 1:100

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Phospho-mTOR (Ser2448) (49F9) Rabbit mAb detects endogenous levels of mTOR protein only when phosphorylated at Ser2448.


Species Reactivity: Human
Species predicted to react based on 100% sequence homology: Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser2448 of human mTOR.

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).


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.
U.S. Patent No. 5,675,063.

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Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific)