The monoclonal antibody BM8 recognises a 125 kD extracellular macrophage membrane molecule, highly restricted to mature macrophage subpopulations residing in tissue. This murine F4/80 glycoprotein contains seven-transmembrane (TM7) regions, which anchor the protein in the cell membrane, and thereby shows similarity in this region to G-protein-coupled receptors. The F4/80 molecule shares overall structural homology to other members of the epidermal growth factor (EGF)-TM7 family. The antigen is detected on tissue fixed macrophages in all organs tested so far (spleen, lymph nodes, thymus, liver, skin). It is also present on Langerhans cells in the skin and Kupffer cells in the liver. It is absent on granulocytes, lymphocytes and trombocytes. The expression of F4/80 increases upon maturation of macrophage precursors in bone marrow and blood as well as in ontogeny.
The monoclonal antibody BM8 is the only macrophage marker that is able to distinguish non-destructive from destructive inflammation processes in the pancreas. Furthermore it is a unique histological marker of the progression from peri-insulitis to beta-cell destruction and diabetes in a mouse diabetes model.
Flow cytometry, Frozen sections, Paraffin sections, Western blot
W: Mouse bone-marrow derived macrophages; non-reduced; ~125 kDa (Ref 1); reduction with 2-mercaptoethanol destroys BM8 antigen.
F: tissue embedded in OCT Tissue Tec; fixed with acetone for 10 min at RT; incubation with 0.02 M sodium azide in PBS containing 0.1 % H2O2 for 10 min at RT to destroy endogenous peroxidase; spleen as positive control.
P: fixation in 10% neutral buffered formalin for 24 h; blocking with non-immunized goat serum; microwaved for 6 min in citrate buffer; splenic macrophages as positive control (Ref 3).
FC: fixed with 1 % paraformaldehyde (Ref 1).
Dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.
Mouse fibroblasts or granulocytes