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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.
  6. Antigen Unmasking Citrate: 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.
  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: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
  9. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  10. Substrate: Vector® NovaRED™ (Vector Laboratories).

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: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; 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 two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in blocking solution 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. 5-15 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with 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 sections with coverslips.

posted February 2010

revised November 2013

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. Hematoxylin (optional).
  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. Methanol/Peroxidase: To prepare, add 10 ml 30% H2O2 to 90 ml methanol. Store at -20°C.
  7. Blocking Solution: 1X TBS/0.3% Triton™ X-100/5% Normal Goat Serum (#5425). To prepare, add 500 µl goat serum and 30 µl Triton™ X-100 to 9.5 ml 1X TBS.
  8. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  9. Substrate: Vector® NovaRED™ (Vector Laboratories).

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 methanol/peroxidase.
  3. Wash sections in wash buffer two times for 5 min.
  4. Block each section with 100–400 µl blocking solution for 1 hr at room temperature.
  5. Remove blocking solution and add 100–400 µl primary antibody diluted in blocking solution 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. 5-15 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with 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 sections with coverslips.

posted February 2010

revised November 2013

protocol id: 326

Product Usage Information

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

5.  Gingras, A.C. et al. (2001) Genes Dev. 15, 807-826.

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

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

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

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


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.