|REACTIVITY||H M R|
|Flow Cytometry (Fixed/Permeabilized)||1:50|
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala139 of human Iba1/AIF-1 protein.
Ionized calcium-binding adaptor molecule 1 (Iba1), also known as allograft inflammatory factor 1 (AIF-1), is an evolutionarily conserved cytoplasmic calcium binding protein containing a central pair of EF-hand calcium binding motifs (1,2). Iba1/AIF-1 was originally cloned from activated macrophages in human atherosclerotic allogenic heart grafts undergoing chronic transplant rejection as well as from rat monocytes (3,4). Its function is not very well understood, but Iba1/AIF-1 expression is upregulated in response to interferon-gamma and, therefore, could modulate macrophage-dependent immune response (3). As an F-actin-binding protein, Iba1/AIF-1 may function to remodel the actin cytoskeleton and contribute to morphological changes that correlate with various microglial/macrophage states (5). Iba1/AIF-1 is also uniquely expressed in cells of monocytic lineage and is, therefore, widely used as a marker for microglia/macrophages in the brain and other tissue (6).
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