Flow Cytometry

Direct immunofluorescence staining of intracellular antigens: Methanol plus Leucoperm Accessory Reagent method

Direct Staining of Intracellular Antigens by Flow Cytometry: Methanol plus Leucoperm Method

Direct Staining of Intracellular Antigens by Flow Cytometry: Methanol plus Leucoperm Method

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Alternative protocol for cell permeabilization required prior to intracellular staining

The detection of intracellular antigens requires a cell permeabilization step prior to staining. This method provides an alternative procedure for use when protocol FC7. “Direct Staining of Intracellular Antigens by Flow Cytometry using Leucoperm does not provide the desired results. This method is particularly suitable for the detection of nuclear antigens, such as PCNA and Ki67. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with the product and batch specific information provided with each vial. A certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques; these are guidelines only and may need to be adjusted for particular applications. Specific methodology for blood appears in [ ] brackets.

Reagents ‎

Leucoperm Accessory Reagent (Product code BUF09). Includes Reagent A (cell fixation agent) and Reagent B (cell permeabilization agent).
Phosphate buffered saline (Product code BUF036A) containing 1% bovine serum albumin (PBS/BSA)
Erythrolyse red blood cell lysing buffer (BUF04).
Anticoagulant (Note: for basic staining any appropriate anticoagulant, such as heparin, EDTA, or acid citrate dextrose, may be used. In some instances specific anticoagulants may be required.)
Optional: 0.5% (w/v) paraformaldehyde in PBS (Note: dissolve on heated stirrer and cool before use)


  1. Harvest cells and determine the total number present. Adjust cell suspension to a concentration of 1 x 107 cells/ml in PBS/BSA.
    [Whole blood samples may also be used. Bio-Rad recommends the use of EDTA anti-coagulant in ‎these circumstances, although satisfactory results may be obtained using heparin or acid-citrate ‎dextrose.]
  2. Add 100 μl of cell suspension [or whole blood] to the appropriate number of test tubes.
  3. If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at 4oC, avoiding direct light.
  4. Wash cells once in 2ml cold (4oC) PBS/BSA and discard the supernatant.
  5. Re-suspend cells in 100 μl cold (2-8oC) Leucoperm Reagent A (cell fixation agent) per 1 x 106 cells. Incubate for 10 minutes at 2-8oC.
  6. Add 500 μl of ice cold absolute methanol, vortex and incubate for 10 minutes at 2-8oC.
  7. Add 3 ml cold (4oC) PBS and centrifuge for 5 minutes at 300-400 g at 4oC.
  8. Remove supernatant and add 100 μl Leucoperm Reagent B (cell permeabilization agent) per 1 x 106 cells. Add the directly conjugated antibody at the vendor-recommended dilution and incubate at 4oC for at least 30 minutes, avoiding direct light.
    [To the blood suspension add 2ml freshly prepared erythrolyse red cell lysing buffer and mix well. Incubate for 10 min at room temperature. Centrifuge at 300-400 g for 5 min and discard the supernatant. Wash with 2ml room temperature PBS/BSA, centrifuge at 300-400 g for 5 min at room temperature and discard the supernatant. Continue to step 9.]
  9. Resuspend cells in 2 ml cold (4oC) PBS and centrifuge at 300-400 g at 4oC. Discard supernatant.
  10. Resuspend cells in 200 μl cold (4oC) PBS for immediate analysis or with 200 μl 0.5% formaldehyde in PBS if required.
  11. Acquire data by flow cytometry. Analyze fixed cells within 24 hours.


  • Phycoerythrin conjugates are not suitable for the detection of cell surface antigens using this method due to damage of the RPE at low temperatures.
  • To avoid unspecific binding, you also need to block Fc receptors on cell types such as spleen cells, with FcR blocking reagents e.g. Bio-Rad’s Mouse Seroblock reagent (BUF041).
  • Appropriate controls should always be carried out, for flow cytometry the following should be considered for inclusion;
    • Isotype controls used to determine if the staining is specific.
    • Unstained cells should always be included in the experimental set-up to monitor autofluorescence.
  • For all multi-color Flow Cytometry experiments it is advisable to include compensation controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating boundaries.