Foxp3 Frequently Asked Questions
For additional information on our Foxp3 products, please see the Foxp3 Knowledge Center
Q: What are the different Foxp3 reagents available for intracellular staining and flow cytometric analysis?
The following reagents are currently available:
- Anti-Mouse/Rat Foxp3 (clone FJK-16s)
- Anti-Human CD4+CD25bright Foxp3 (clone PCH101)
- Anti-Mouse Foxp3 (clone NRRF-30)
- Anti-Human Foxp3 (236A/E7)
Q: What is the difference between the Foxp3 antibodies?
The antibodies recognize different epitopes in the Foxp3 protein. Each epitope is listed on the individual data sheet. All antibodies recognize different regions of the protein allowing them to be used together to confirm staining of the same cells/tissue distribution, as well as studying different isoforms or modifications.
Q: Why are the antibodies sold as a set with buffers? Can I purchase the antibodies separately?
We have optimized the buffers and protocols for Foxp3 staining. Variations from these protocols and buffers will yield different results (or no results). It is critical that you use the appropriate buffers and protocol so that we can provide technical support should you have any questions. We would prefer that you purchase the kit, particularly when using the antibodies for the first time. The antibodies are also sold separately.
Q: After the fixation/permeabilization step the cells are gone. Where did they go?
The Fixation/Permeabilization is not a lysis step but rather a fixation step that also permeabilizes. It was optimized for staining with freshly isolated PBMCs or mouse splenocytes. The cells become smaller and are more translucent giving the appearance that the pellet is greatly diminished. But if the pellet is resuspended, the same number of cells will be present as confirmed by observing the cells under the microscope. Because of the change in size, the voltages have to be adjusted to "find" the cells. We have tested many other buffers and have not found any commercially available buffer to give the same robust and reproducible results as the Foxp3 buffer we supply.
Q: Are you able to stain surface antigens using these antibodies?
When using the Foxp3 antibodies, we have not had any problems with surface staining. Because the staining conditions are mild, we do not see any decrease in the staining intensity due to the protocol. We have not tested all antibodies, so there is the possibility that the antigen recognized by some antibody clones may be destroyed/modified during the fixation step such that the antibody can no longer bind.
Q: At what stage in the protocol do you surface stain for proteins such as CD25 and CD4?
Like traditional intracellular staining protocols, we have tested surface staining before fixation. It is possible that many of these antibodies will also work post fixation but this should be tried by the researcher for individual antibody clones. For example CD4 (RPA-T4) can be stained either at the beginning of the protocol or concurrently with PCH101 (Foxp3).
Q: Can the staining protocol be shortened? Can I fix/perm for more than overnight?
We have found that for the anti-mouse/rat Foxp3 staining set (FJK-16s and NRRF-30), you can decrease the Fix/Perm step to 30 minutes. Less than that can reduced the amount of staining observed. If left longer than overnight in the fixation/permeabilization solution, we see reduced staining. The results from 0.5-18 hours have yielded equivalent results both in the percentage of positive cells and the fluorescence intensity of staining. The anti-human foxp3 antibodies are more sensitive to the fix/perm time. For human we only recommend using 30-60 minutes with the fixation/permeabilization solution. We do not recommend trying to fix the cells prior to beginning the Foxp3 protocol, as this will dramatically affect your results. Please see question below regarding extending the time for PCH101 and 236A/E7.
Q: I can't get on the cytometer to read my samples. What can I do?
For the mouse/rat foxp3 staining there is a range of time for the fix/perm incubation. For the human foxp3 staining we do not recommend varying the fix/perm incubation. Instead we have found that you can complete the staining and put the cells in Flow Stain Buffer at 4C overnight in the dark and analyze the next morning. For human foxp3, we do see a decrease in the fluorescent intensity but the population does remain.
Q: Can these antibodies be used for IHC?
We have reports of PCH101, 236A/E7, and FJK-16s staining frozen tissue sections using both acetone and PFA. For 2-color staining, the conditions may need to be determined for all antibodies used. For example, CD4 staining may require acetone fixation. We have information from the literature that the clones PCH101, 236A/E7, and FJK-16s will work on paraffin embedded tissues. Please refer to the product datasheet for the references regarding each of these clones. For a detailed protocol, please contact Technical Support.
Q: What happened to hFOXY?
We have found that researchers prefer the PCH101 (Anti-Human Foxp3 antibody) Staining Set because the protocol is quite fast and mild. All surface labeling tried has been retained throughout the procedure.
Q: Which antibodies can I use for western blotting?
The following are our recommendations:
- Anti-Mouse/Rat Foxp3- FJK-16s
- Anti-Human Foxp3 PCH101
- Anti-Mouse/Human Foxp3 eBio7979
Q: I want to obtain good results, so which format of Foxp3 should I order?
Our recommendation is to use the Foxp3 antibodies in PE or APC as they are brighter than FITC. Furthermore we prefer CD4 in FITC since the expression levels are high enough to use with this fluorochrome. We then use CD25 in APC. Please see our Treg staining Kits for prebundled kits with optimal fluorochrome combinations (88-8999 and 88-8111).