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

Name: F4/80 Antibody

Clone: BM8, Rat anti-Mouse

Supplier: Caltag Labs (Invitrogen)

Catalog Number: MF48000

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.

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: 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