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CD4 Antibody Staining Protocol for Immunohistochemistry
Description: CD4, a single chain transmembrane glycoprotein, is found on a T cell subset (helper/inducer) representing 45% of peripheral blood lymphocytes. It is also present on 80% of thymocytes and at a lower level on monocytes. It is involved in recognition of antigen presented along with MHC class II by APCs. It serves as receptor for HIV.
Primary Antibody
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Name: CD4 Antibody |
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Clone: 1F6, Mouse monoclonal |
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Supplier: Lab Vision |
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Catalog Number: MS-392-S |
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Dilution: 1:10 - 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: 60 minutes/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). Other buffers (citrate, Tris-EDTA, etc.) has been tested but the results were NOT satisfactory. |
| Heat/Cool Temperature: 95 ºC - 100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods - Recommended detection method. |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Gill's Hematoxylin or Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cell membrane |
| Images: Search image |
Additional Information:
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Species Reactivity: Human |
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Fixation: Formalin fixed, paraffin embedded sections |
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Positive Control: Human tonsil (T-cells) |
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Negative Control: Omit primary antibody, isotype control or absorption control |
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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: