The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 10 µg/ml.
Use 3µg for 106 cells. ab18536-Rat monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
Use 2µg for 106 cells.
ab25235 can be used for blocking of Fc gamma receptors.
RelevanceIn mouse only the transmembrane form of CD16 is reported to exist, which is expressed on macrophages, NK cells, granulocytes, myeloid precursors, and subpopulations of T lymphocytes. CD32 is primarily expressed on cells of the myeloid lineage and also on mature B lymphocytes. The lymphocyte Fc gamma receptors recognize the Fc portion of IgG, presented either as immune complexes or as free antibody. The different classes of receptors are distinct because of varying size, tissue distribution and affinity for IgGs. The Fc type II receptor is expressed on a wide variety of cells including B cells, hematopoietic cells, monocyte/macrophages, neutrophils, platelets, Langerhans cells, eosinophils, basophils, trophoblasts, and endothelial cells of the placenta. The Fc gamma type III receptors are higher affinity than the type II and are expressed on macrophages, NK cells and neutrophils. Both types of receptors can be expressed on the same cell and in varying ratios. The receptors are constitutively expressed, although cytokines and lymphokines can modulate their expression.
Cellular localizationCD32: Type I membrane protein.
CD16: Attached to the membrane by a GPI anchor. Exists also as a soluble receptor, produced by a proteolytic cleavage.
ICC/IF image of ab25235 stained RAW246.7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab25235, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab98420, DyLight® 488 goat anti-rat IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.