We do two rat antiMouse primaries for immunofluorescent staining routinely, and have done triple staining also, without problems.
You must do sequential staining with the primaries.
Our favorite method is to use one biotinylated antibody, and that one is used after the first primary/secondary application. For immunofluorescent work we do NOT use BSA or any protein carriers in our buffers, but do add 0.025% Tween 20 for sheeting action. Rinsing well is critical for IFA work. Spin all fluorophores after dilution and just before application to get rid of any fluorophore labelled protein aggregates (glowing garbage!).
Our frozen sections are air dried overnight at RT, and fixed the next day - if you use acetone, 10 min at 4C, air dry then proceed to buffer after getting rid of acetone fumes.
Normal serum block 5% donkey (or goat depending on host of secondary) serum 30 min RT
streptavidin/biotin block is needed. (Vector kit)
1. FOXP3 primary rat antimouse monoclonal diluted in 5% donkey serum, this may not need overnight incubation unless you have determined you need overnight for this primary.
2. Donkey antiRat F(ab')2 frag of IgG - Rhodamine Red X, adsorbed to mouse tissue 1:300 or so diluted in 5% donkey serum. 30 min RT (Jackson Immunoresearch for this antibody)
For secondary antibody, you can purchase a different one from Molecular Probes as you did, and the Alexa 594 it will have better spectral separation from the 488. The seconday should be adsored to the mouse. If the secondary is made in goat then use goat serum instead.
3. Rat serum, 5% for 30 minutes in order to ensure the secondary antibody is bound to rat, and does NOT recognize the next rat antimouse primary, even if it is biotinylated. Rinse well.
4. CD4-biotinylated (BD Biosciences) 1:500 in 5% donkey serum/1.25% mouse serum 30 minutes at RT
5. Strepavidin-488 diluted in buffer with Tween, NO SERUM with this step. Molecular Probes gives a dilution range up to 1:1000, and this will work, incubate in dark for 30 min, RT.
Rinse well with pure buffer and mount coverslip with Prolong Gold antifade reagent, ready to use from Molecular Probes
The denaturing solution is NOT needed when doing this method.
Using a biotinylated primary is a good way to avoid cross reaction to a secondary antibody previously used, but the rat serum block is critical. =20
If you want to see a photograph of this type of staining method, I will be happy to attach on to you privately, but will need your email address.
I think the problem is that you are blocking with rat serum.
You wouldn't block with rat serum before your first primary, and you shouldn't for your second primary since both are rat antibodies.
There are several ways you could try to accomplish dual staining of 2 rat antibodies. I think the easiest, since you are doing fluorescent on frozens, is use BD's rat x mouse CD4 FITC (green cytoplasm). It works very well. Ebioscience has AF conjugated primaries, so you should be able to get rat x FOXP3 AF555 or AF594 (red nucleus). Then you could just cocktail them and do single step staining.
Or you could try Probes (Invitrogen's) Zenon labeling kits, and directly conjugate your rat antibodies yourself. (I don't know if they have kits for rat primaries though)
Thirdly, you could try following your protocol for the first primary, adding Biocare Medicals' Denaturing solution to seal the antigen site (since the first is cytoplasmic) then the second would have access to the nuclear site.
1. 5% normal goat serum block 20 min
2. 1* antibody CD4 1:750 in diluent O/N 4C
3. Seconday AF594 GT anti-rat 1:1k in 1xTBS 1h RT
4. Biocare Medicals' Denaturing solution (follow manuf instructions)
5. Serum block: 5% normal DONKEY serum 30 min
6. 1* antibody FOXP3 1:100 in diluent 1h RT7. Secondary AF488 Donkey anti-rat in TBS 1h RT
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