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CD20 Antibody Staining Protocol for Immunohistochemistry
Description: This antibody can be used to identify B-cells in normal and neoplastic tissues. The L26 antibody reacts with two non-covalently bonded components weighing 33 kDa and 30 kDa present in the majority of B-cells. Positive staining with L26 occurs on the cytoplasmic side of the cell membrane. Positive staining was demonstrated using the L26 antibody and COS-1 cells transfected with cDNA encoding for the CD20 antigen. The L26 antibody detects an epitope which survives tissue processing, and is suitable for use on routinely fixed, paraffin-embedded tissues. The CD20 antigen is expressed in most B-cells present in peripheral blood and lymphoid tissue. The antigen is also found in most non-Hodgkin’s lymphomas of B-cell lineage. Cross-reactivity has not been observed with normal or malignant non-lymphoid cells except for: L26-positive dendritic cells in the medulla of the thymus and thymomas.
Primary Antibody
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Name: CD20 Antibody |
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Clone: L26, Mouse anti-Human |
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Supplier: Dako |
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Catalog Number: M0755 |
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Dilution: 1:300 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: Cytoplasmic side of the cell membrane |
| Images: Search image |
Additional Information:
| Tissue Type: Lymph node, tonsil, spleen, thymus |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Lymph node, tonsil, spleen, 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 |
References: