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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
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Name: Troponin T Antibody |
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Clone: JLT-12, Mouse anti-Rabbit |
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Supplier: Sigma-Aldrich |
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Catalog Number: T6277 |
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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. |
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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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
References: