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Fix cells before intracellular staining to ensure stability of soluble antigens or antigens with a short half-life (see the special recommendations below for exceptions). This retains the target protein in the original cellular location.
Detecting intracellular antigens requires cell permeabilization before staining. Antibodies should be prepared in permeabilization buffer to ensure the cells remain permeable. When gating on cell populations, the light scatter profiles of the cells on the flow cytometer will change considerably after permeabilization. NB cell surface staining should be performed prior to fixation.
Several methods are available for cell fixation and permeabilization:
Formaldehyde followed by detergent
Fix in 0.01% formaldehyde for 10–15 min, then disrupt membranes using one of the following detergents:
Formaldehyde (0.01%) followed by methanol
Methanol followed by detergent
Acetone fixation and permeabilization
Plastic tubes are not suitable for use with acetone.
Antigens close to the plasma membrane and soluble cytoplasmic antigens will require mild cell permeabilization without fixation.
Cytoskeletal, viral and some enzyme antigens usually give optimal results when fixed with a high concentration of acetone, alcohol or formaldehyde.
Antigens in cytoplasmic organelles and granules will require a fixation and permeabilization method depending on the antigen. The epitope needs to remain accessible.
Prepare antibodies in permeabilization buffer to ensure the cells remain permeable.
Detecting secreted proteins can be challenging because they may degrade rapidly. Use Brefaldin A or other compounds that prevent protein release from the Golgi apparatus, enabling the detection of cells expressing the protein.