[Histonet] RE: CD25 on murine tissue woes

Mathew DeGutes m-degutes <@t> northwestern.edu
Thu Jul 21 15:00:54 CDT 2005

If you want all the nitty gritty details.  General protocol is this

1. Cut sections.  Let dry and store at -80c until use.
2. Let sections return to room temperature and then fix. (Have tried Acetone 10 min -20c, 2%PFA 5
min room temp)
3. Wash 3x5 PBS
4. Wash 5 min in 3% H2O2 (have tried without this step)
5. Wash 3x5 PBS
6. Encircle and Avidin Block 10 min. (avidin biotin blocks are dependent on whether a biotinylated
antibody is used).
7. Wash off Avidin in PBS
8. Biotin block 10 min
10. Aspirate Biotin block and add Block (have tried various serum at 10% and TNB, either with
CD16/32 added at 1:50) for 30 min minimum.
11. Aspirate block and add Primary ab (have tried both 7D4 biotinylated anti-CD25 from Pharmingen
and a PC61 Fitc-conjugated anti-CD25 from eBioscience at concentrations from 1:25 to 1:500) diluted 
in TNB or a 2% serum + .2% Triton in PBS buffer for 1 hour at room temp (have also tried overnight
at 4c, but generally do the 1hour room temp).
12. Wash 3x5 PBS
13. Add 2ndary ab (Either Streptavidin-HRP or Anti-Fitc HRP) 1:100 in staining buffer from step 11
and inc for 30 min.
14. Wash 3x5 PBS
15. Use TSA Cy3 or Fluorescein kit.  Dilute 1:100 in diluent and develop 5 minutes.
16. Wash 3x5 PBS
17. Mount with Vectashield with DAPI and coverslip

Now we have used these 2ndaries and TSA kits extensively and have found them to work very well on
any number of antigens.  As I said before I haven't attempted to make it work with a direct.  I
come back and amplify with anti-fitc-hrp and the TSA kit.  Also we are generally working in SJL
mouse strains.  I would really like to get this to work to go alongside FoxP3 staining for regs.  I 
realize that regs are supposedly lower in SJL strains, but I have attempted this on Naive BalbC
spleens as well and also have attempted on spleens from mice that were given injections of CD25+
CD4+ Tcells and so should have a far greater number of cells that are CD25+ (whether these cells
will actually show up in anything other than flow I am not sure).

You Wrote:
Could you provide more details on HOW you are doing the staining, both as 
IHC and or immunofluorescence.  If you are trying to do the primary 
antibody directly conjugated to FITC on a tissue section, you may not get 
positive results - this is not uncommon with some of this type conjugate as 
a direct IFA stain.  I can't make it work with CD4-FITC or CD8-FITC.

As for biotinylated antibodies (primaries) one can do either IHC and IFA - 
we do this on many other rat antimouse primaries, CD markers - so provide 
concentrations, times, etc, etc - all those good things.

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