Troponin T (TnT) Antibody Staining Protocol for Immunohistochemistry


Description: Troponin T (TnT) is a member of the troponin complex -- consisting of troponins I, C, and T -- responsible for binding tropomyosin to regulate calcium binding and contractility of vertebrate striated muscle. There are three known isoforms for troponin T: fast skeletal, slow skeletal, and cardiac. The cardiac isoform of troponin T (cTnT) exists in types I (cTnT-I) and II (cTnT-II).


Primary Antibody

Name: Troponin T Antibody

Clone: JLT-12, Mouse anti-Rabbit

Supplier: Sigma-Aldrich

Catalog Number: T6277

Dilution: 1:100 - 1:200 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:
Species Reactivity: Rabbit, rat, bovine, chicken
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections (no antigen retrieval needed)
Positive Control: Cardiac or skeletal muscle. Rat cardiac muscle works well
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