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CD40 Antibody Staining Protocol for Immunohistochemistry
Description: The 3/23 antibody reacts with CD40, a 40-50–kDa glycoprotein expressed on B lymphocytes, follicular dendritic cells, bone marrow-derived dendritic cells, thymic epithelium, and interdigitating cells in the T-cell zones of secondary lymphoid organs. The CD40 molecule has a central role in B cell growth and differentiation. Ligation of CD40 is reported to be an important signal in B-cell survival. Costimulation with anti-CD40 and IL-4 rescues B cells from apoptosis induced by extensive cross-linking of surface IgM or IgD. Furthermore, signaling through CD40 in combination with IL-4 reportedly induces immunoglobulin isotype switching and secretion of IgE by human B cells. Ligation of CD40 with the 3/23 antibody can induce splenic B cells to express the costimulatory molecule CD86 (B7-2).
Primary Antibody
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Name: CD40 Antibody |
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Clone: Rat anti-Mouse |
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Supplier: Pharmingen |
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Catalog Number: 550285 |
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Dilution: 1:50 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: Incubator |
| Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101) |
| Heat/Cool Temperature: 37 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 |
| Images: Search image |
Additional Information:
| Tissue Type: Spleen, thymus |
| Fixation: Zinc fixative fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Spleen, thymus, B cells, dendritic cells |
| 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 on acetone fixed frozen sections |
References: