Direct Immunofluorescence Staining of Intracellular Antigens Using Antibodies Labeled with StarBright Dyes

Direct immunofluorescence staining of intracellular antigens using antibodies labeled with StarBright Dyes

Direct Immunofluorescence Staining of Intracellular Antigens Using Antibodies Labeled with StarBright Dyes

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For use with antibodies labeled to StarBright™ Dyes using TrailBlazer™ Tag and TrailBlazer StarBright Dye Label Kits when intracellular staining is required.

To detect intracellular targets, such as cytoplasmic proteins, cytokines, and nuclear proteins, cell permeabilization is required to allow the antibody to interact with its target antigen.

This protocol describes intracellular staining using the eBioscience Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific Inc., catalog 00-5523-00), which fixes and permeabilizes cells, and includes a blocking step, followed by staining in a buffer containing BD Horizon Brilliant Stain Buffer (BD Biosciences, 563794). Alternative intracellular staining protocols for antibodies conjugated to non-StarBright Dye fluorophores include FC9 for the staining of cytokines in whole blood, FC10 for the digitonin method, and FC11 for the paraformaldehyde/saponin method.

This protocol is a guideline only and may need to be optimized for particular cell types or antibodies. 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 antibody vial. A certain level of technical skill is required for the successful design and implementation of these techniques.

Reagents

  • Phosphate buffered saline (10x, Bio-Rad Laboratories, Inc., BUF036A) containing 1% bovine serum albumin (PBS/BSA)
  • Phosphate buffered saline (PBS) (10x, Bio-Rad, BUF036A)
  • Staining Buffer (Bio-Rad, BUF073): Required when staining cell surface antigens
  • eBioscience Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, 00-5523-00) containing:
    • Fixation/permeabilization concentrate
    • Fixation/permeabilization diluent
    • Permeabilization buffer (10x)
  • dH2O
  • Blocking reagent; e.g. Human Seroblock (Bio-Rad, BUF070), Mouse Seroblock FcR (Bio-Rad, BUF041) or serum
  • BD Horizon Brilliant Stain Buffer (BD Biosciences, 563794)
  • Optional: Tween 20
  • Optional: Fixable live/dead dye

Method

  1. Prepare cells appropriately. For information on preparation of cells from tissue culture cell lines, tissues, and isolation of peripheral blood mononuclear cells from whole blood, refer to protocols FC1, FC2, or FC3. Resuspend cells at a concentration of 1 x 107 cells/ml in Staining Buffer (Bio-Rad, BUF073) at 4oC.
  2. Aliquot 100 μl of the cell suspension into as many 5 ml tubes as required. 
    Optional: Perform staining of cell surface antigens and associated wash steps. Refer to protocols FC4 and FC22 for more details.
  3. Wash cells once in 2 ml cold (4oC) PBS/BSA and discard the supernatant.
  4. Prepare Foxp3 fixation/permeabilization working solution by mixing 1 part of fixation/permeabilization concentrate with 3 parts of fixation/permeabilization diluent.
  5. Add 1 ml of Foxp3 fixation/permeabilization working solution to each tube and vortex, ensure the pellet is fully dissociated. Incubate in the dark at room temperature (RT) for 30 min.
  6. Prepare 1x Foxp3 permeabilization buffer by mixing 1 part of permeabilization buffer (10x) with 9 parts of dH2O.
  7. Add 2 ml of 1x permeabilization buffer to each tube and centrifuge for 5 min at 400–600 x g at RT. Discard supernatant.
  8. Repeat step 7.
  9. Resuspend cells in residual volume of permeabilization buffer, typically 100 µl, and block for 15 min at RT with chosen blocking reagent. See notes section for common blocking reagents.
  10. Add Brilliant Stain Buffer to a total volume of 20–50% and add the StarBright Dye–labeled antibody at the dilution determined by a titration experiment. Mix well and incubate for 60 min at RT or 18 hr (overnight) at 4oC, avoiding direct light.
  11. Add 2 ml of 1x permeabilization buffer to each tube, centrifuge at 400–600 x g for 5 min at RT. Discard the supernatant.
  12. Repeat step 11.
  13. Resuspend cells in 200 μl cold (4oC) PBS and store in the dark at 4oC.
  14. Acquire samples on a flow cytometer. Analyze fixed cells within 48 hr.

Notes

  • A blocking reagent is strongly recommended to reduce background staining. The most effective blocking reagent is cell- and antibody-dependent. Typical blocking reagents for human cells are 5 µl Human Seroblock (Bio-Rad, BUF070), and for mouse cells, 1 µl of Mouse Seroblock (Bio-Rad, BUF041). Serum is also an effective blocking reagent when used at a concentration of 3–10%
  • The addition of 0.1% Tween 20 during antibody incubation may also reduce any non-specific background
  • Different incubation times have been effective for intracellular staining using StarBright Dye-conjugated antibodies from 1 hr at RT to 18 hr (overnight) at 4oC. Use the most appropriate protocol for your experiment
  • Antibodies labeled using a TrailBlazer Tag and the TrailBlazer StarBright Dye Label Kit typically stain optimally at 5 µl per test for 1 hr at RT. A lower dilution is required for overnight staining. It is best practice to titrate all antibodies in your experiment using the same conditions as in the final experiment. Further information regarding titration best practices is available on the Bio-Rad website
  • Including a viability dye staining step prior to Step 4 is recommended to allow gating on live cells during analysis. A fixable dye is required for intracellular staining, such as VivaFix Cell Viability Assay (Bio-Rad, 135111)