Anti foxp3

Sections of formalin-fixed paraffin-embedded hearts and frozen spleens were used for detection of CD3 and Foxp3 expression by immunofluorescence. First, paraffin-embedded sections were deparaffinized and submitted to a heat-induced antigen retrieval step by incubation in citrate buffer (pH 6.0). Then, sections were incubated overnight with the following primary antibodies: anti-CD3 (1:400; BD Biosciences) and anti-Foxp3 (1:400; Dako, Glostrup, Denmark). On the following day, secondary antibodies anti-goat IgG Alexa Fluor 488 (1:600; Molecular Probes) or anti-rabbit IgG Alexa Fluor 568 conjugated (1:100; Molecular Probes), diluted in 1% BSA in PBS, were added. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; VectaShield Hard Set mounting medium with DAPI H-1500; Vector Laboratories, Burlingame, CA). Images were analyzed using a confocal laser scanning microscope A1R (Nikon, Tokyo, Japan) and Image-Pro Plus version 7.01 (Media Cybernetics, Rockville, MD). Quantifications of CD3⁺/Foxp3⁺ cells percental area were performed in 10 fields randomly captured under x400 magnification, using Image-Pro Plus v.7.0.