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

Name: F4/80 Antibody

Clone: CI:A3-1, Rat anti-Mouse

Supplier: Caltag Labs (Invitrogen)

Catalog Number: RM290

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: 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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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


Not tested on frozen sections