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F4/80 Antibody Staining Protocol for Immunohistochemistry
Description: The CI: A3-1 monoclonal antibody (mAb) reacts with the F4/80 antigen which is a macrophage - restricted cell surface glycoprotein. Applications of the CI: A3-1 mAb include immunohistochemistry and immunostaining for flow cytometry.
Primary Antibody
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Name: F4/80 Antibody |
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Clone: CI:A3-1, Rat anti-Mouse |
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Supplier: Caltag Labs (Invitrogen) |
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Catalog Number: RM290 |
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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: 60 minutes/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: Gill's Hematoxylin or Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Membrane/cytoplasmic |
| Images: Search image |
Additional Information:
| Species Reactivity: Mouse |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Spleen |
| 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 tested on frozen sections |
References: