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CD8 Antibody Staining Protocol for Immunohistochemistry
Description: The CD8 molecule is composed of two chains and has a molecular weight of 32kD. It is found on a T cell subset of normal cytotoxic/suppressor cells which make up approximately 20 to 35 percent of human peripheral blood lymphocytes. The CD8 antigen is reported to be detected on natural killer cells, 80 percent of thymocytes, on a subpopulation of 30 percent of peripheral blood null cells and 15 to 30 percent of bone marrow cells.
Primary Antibody
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Name: CD8, T Cell Antibody |
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Clone: 4B11, Mouse anti-Human |
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Supplier: Novocastra Labs |
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Catalog Number: NCL-CD8-4B11 |
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Dilution: 1:20 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: 60min/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: Membrane of T Cells |
| Images: Search image |
Additional Information:
| Tissue Type: Tonsil |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Tonsil, lymph node, thymus |
| Negative Control: Omit primary antibody, isotype control or 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 |
Notes:
Not need to perform antigen retrieval for acetone fixed frozen sections |
References: