For immunofluorescent staining, the suggested use of this reagent is 5 µl per 10⁶ cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application. Each lot of this FOXP3 antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.
 

* Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm.
** Alexa Fluor® is a registered trademark of Molecular Probes, Inc. Alexa Fluor® dye antibody conjugates are sold under license from Molecular Probes, Inc. for research use only, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents.

Materials Provided:
1. Alexa Fluor® 488 anti-mouse FOXP3, 25 µg (Cat. No. 126405)
2. Alexa Fluor® 488 Rat IgG2b, k isotype control, 25 µg (Cat. No. 400625)
3. FOXP3 Fix/Perm buffer set, 100 tests (Cat. No. 421403)
Materials not included:
Cell Staining Buffer (Cat. No. 420201)
Anti-mouse CD4 APC/CD25 PE cocktail (121301)

Surface Staining & FOXP3 Buffer Preparation:

Centrifugation steps: perform at 250Xg for 5min
Incubation steps: perform at room temperature.

1. Perform cell surface staining if necessary (See protocol: Cell Surface Immunofluorescence Staining Protocol).
2. Prepare 1X buffer solutions: The FOXP3 Fix/Perm buffer (4X) must be freshly diluted by diluting one (1) part FOPX3 Fix/Perm buffer (4X) with three (3) parts PBS. The FOXP3 Perm buffer (10X) should be diluted by diluting one (1) part FOXP3 Perm buffer (10X) with nine (9) parts of PBS.

NOTE: The FOXP3 Perm buffer (10X) may have crystalization or precipitation observed when it is stored at 2-8°C, however, it is normal and does not affect the buffer performance. If there is a heavy precipitation observed after diluting to 1X working solution, it may be clarified by filtering.
Caution: The FOXP3 Fix/Perm buffer contains paraformaldehyde, which is toxigenic and mutagenic. Please handle with caution and wear gloves, lab coat and necessary protection to avoid direct body contact.

FOXP3 Intracellular Staining Procedures:
3. Add 1 ml of 1X BioLegend's FOXP3 Fix/Perm solution to each tube, vortex and incubate in the dark for 20 minutes, then centrifuge and remove the supernatant. The cell pellet will now be translucent and difficult to see; take care not to dislodge and accidentally aspirate cells at all later stages of staining protocol.
4. Wash: resuspend cells in cell staining buffer (Cat. No. 420201), centrifuge then discard the supernatant.
5. Wash: resuspend in 1ml 1X BioLegend's FOXP3 Perm buffer, centrifuge then discard the supernatant.
6. Resuspend cells in 1ml 1X BioLegend's FOXP3 Perm buffer, incubate in the dark for 15 minutes, centrifuge then discard the supernatant. Resuspend the pellet in 100 µl of 1X BioLegend's FOXP3 Perm buffer.
7. Add 5 μl per tube of Alexa Fluor ® 488 anti-mouse/rat/human FOXP3 antibody, or Alexa Fluor ® 488 mouse IgG2b, κ isotype control to the appropriate tubes, incubate in the dark for 30 minutes.
8. Wash twice with cell staining buffer (see step 4) then resuspend in 0.5ml cell staining buffer . Analyze with flow cytometer using appropriate instrument settings.

** Alexa Fluor® is a registered trademark of Molecular Probes, Inc. Alexa Fluor® dye antibody conjugates are sold under license from Molecular Probes, Inc. for research use only, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents.

1. Ono M, et al.:Nature 2007 446:685
2. Hori S, et al. 2003. Science 299:1057
3. Fontenot JD, et al. 2003 Nature Immunol 4:330
4. Fallarino F, et al. 2009. J. Immunol. 183:6033. PubMed
5. Barber A, et al. 2009 J. Immunol. 183:6939. PubMed
6. Nakashima H, et al. 2010. J. Immunol. 184:4637. PubMed