Protocol: Direct Immunofluorescence Staining of Intracellular Antigens: The Paraformaldehyde/Saponin Method

Reagents

• Phosphate Buffered Saline 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• 4% (w/v) paraformaldehyde in PBS
  Note: Dissolve paraformaldehyde on a heated stirrer and cool before use.
• 0.1% (w/v) saponin in PBS
• Required when staining cell surface antigens:
  • Staining Buffer (BUF073).
• Blocking reagent: Human Seroblock (BUF070) or Mouse Seroblock FcR (BUF041)
• Fixable Live/dead dye
• Optional: Fixation Buffer (BUF071)

Method

1. Prepare cells appropriately and adjust cell suspension to a concentration of 1 x 10⁷ cells/mL with cold (4ᵒC) PBS/BSA.
2. Add 100 μL of the cell suspension into as many 5 mL tubes as required.
3. Optional: Perform staining of cell surface antigens and associated wash steps. Refer to protocol FC4 for more details.
4. Add 3 mL cold (4ᵒC) PBS/BSA, centrifuge at 300–400 x g and 4ᵒC for 5 min, and discard supernatant.
5. Resuspend cells in 100 μL cold (4ᵒC) 4% paraformaldehyde and incubate for 20 min at room temperature (RT).
6. Add 3 mL PBS/BSA, centrifuge for 5 min at 300–400 x g, and discard supernatant.
7. Resuspend cells in 100 μL cold (4ᵒC) 0.1% saponin and incubate for 15 min at RT.
8. Add the directly conjugated antibody at the dilution determined from a titration experiment or the vendor-recommended dilution and incubate for at least 30 min at 4ᵒC while avoiding direct light.
9. Add 3 mL 0.1% saponin, centrifuge at 300–400 x g for 5 min, and discard supernatant.
10. Repeat step 9.
11. Optional: Fix cells by resuspending in 200 µL Fixation Buffer (BUF071) and incubate for 20 min at RT in the dark, centrifuge at 300–400 x g for 5 min, and discard supernatant.
12. Resuspend cells in 200 μL cold (4ᵒC) PBS and store in the dark at 4ᵒC.
13. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hr.

Notes

• The effect of saponin permeabilization is reversible; therefore, it must be present in all washes to effectively remove any unbound antibody from inside of the cell
• It is best practice to block Fc receptors prior to antibody staining to avoid nonspecific binding. We recommend using Bio-Rad's Human Seroblock (BUF070) for blocking human cells and Bio-Rad's Mouse Seroblock FcR (BUF041) for blocking mouse cells
• To allow gating on live cells during analysis, a viability dye staining step prior to step 5 is recommended. A fixable dye such as VivaFix 353/442 Cell Viability Assay (1351111) is required for intracellular staining
• All antibodies should be titrated prior to use to ensure the optimal concentration is used