Phospho-mTOR (Ser2448) Antibody Staining Protocol for Immunohistochemistry


Description: The mammalian target of rapamycin mTOR, also know as FRAP or RAFT(1–3), is a Ser/Thr protein kinase. mTOR acts as a sensor for ATP and amino acids, balancing the availability of nutrients and cell growth (4,5). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal (6), and transmits a positive signal to p70 S6 kinase, and participates in the inactivation of the eIF4E inhibitor, 4E-BP1. 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 cellular growth and homeostasis, and its regulation is frequently altered in tumors. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy.


Primary Antibody

Name: Phospho-mTOR (Ser2448) Antibody

Clone: 49F9, Rabbit monoclonal

Supplier: Cell Signaling Technology

Catalog Number: 2976

Dilution: 1:50 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Cytoplasmic
Images: Search image

Additional Information:
Tissue Type: Human breast carcinoma, colon carcinoma, bladder carcinoma, prostate carcinoma.
Fixation: Formalin fixed paraffin sections, or acetone-fixed frozen sections (no antigen retrieval needed)
Positive Control: Human breast carcinoma, colon carcinoma, bladder carcinoma, prostate carcinoma.
Negative Control: Omit primary antibody, isotype control, absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary