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F4/80 Antibody Staining Protocol for Immunohistochemistry
Description: The BM8 monoclonal antibody (mAb) reacts with the mouse F4/80 which is a macrophage-restricted cell surface glycoprotein. In addition to being useful for immunostaining for flow cytometry, other reported applications for the BM8 mAb include immunohistochemistry of acetone-fixed frozen sections and formalin-fixed paraffin-embedded sections.
Primary Antibody
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Name: F4/80 Antibody |
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Clone: BM8, Rat anti-Mouse |
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Supplier: Caltag Labs (Invitrogen) |
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Catalog Number: MF48000 |
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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: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) or IHC-TekTM Proteinase K Solution (Cat# IW-1101). |
| 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: 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, or acetone fixed frozen sections. |
| Positive Control: Spleen, skin, bladder. |
| 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:
Kristy M. Greeneltch, et al (2007) Host Immunosurveillance Controls Tumor Growth via IFN Regulartory Factor-8-Dependent Mechanisms. Cancer Res 67: (21), 10406-10416. PubMed Abstract