Detection of intracellular antigens by flow cytometry poses unique challenges due to the requirement to access the internal epitope of interest. Usually this means employing some type of cross-linking fixative followed by permeabilization using detergent or alcohol. Reagents can be prepared and tested in-house, or alternately, there are numerous commercial preparations available for the cell fix/perm. Any given test system should be evaluated for optimal performance using appropriate controls (see “Technical Considerations”). Attached is an example of an optimized test system for detection of intracellular antigen using a formaldehyde/methanol fixation and permeabilization combination.
For a complete review of intracellular antigen protocol considerations, see Jacobberger, JW: Flow cytometric analysis of intracellular epitopes. In: Immunophenotyping, eds. Stewart CC and Nicholson JKA, Cytometric Cellular Analysis Series, series eds. Robinson JP and Babcock G, pp. 361-405, 2000.
Technical Considerations: All of the Technical Considerations listed for Cell Surface Antigen Detection apply here with a few additional crucial considerations:
- Choice of fixation/permeabilization: There is an optimum choice of method for the detection of the desired epitope, i.e. one that will allow greatest access of the antibody to the epitope, but it must be determined experimentally. An epitope detected as dim staining by one method may exhibit superior signal-to-noise by another.
- Concentration of cross-linking fixative: For an “in-house” preparation of formaldehyde/paraformaldehyde, we recommend titering the concentration of formaldehyde used (test each concentration with a consistent permeabilization) to check the changes in accessibility to the epitope of interest.
- Use of serum for blocking of background staining: Detailed in the Cell Surface Antigen Detection section, this becomes crucial in intracellular antigen detection as background binding tends to be more problematic.
- Assay controls: Crucial to evaluating if the staining obtained is “real”. It may be worth inquiring of the manufacturer i.e.: the specificity of the antibody as assessed by Western blot (or do yourself). Also consider using a permeabilization control (see core personnel for options).