Immunofluorescent Visualization of T-cell and IFN-γ Responses

4get mice were injected with 1.33 μg anti-CD3 mAb (2C11; BD Biosciences) intravenously in PBS via the tail vein. After 90 min, the spleens were harvested, incubated at 4°C for 2 h in 4% paraformaldehyde, rinsed overnight in PBS, and frozen in OCT embedding compound. 7-μm sections were cut with a cryomicrotome (Leica) and placed onto charged glass slides (Fisher Scientific). Endogenous peroxidase activity was quenched in 1% H₂O₂ and 0.1% azide for 1 h, followed by Fc-block (BD Biosciences) with 1% mouse and rat serum, and avidin and biotin (Vector Laboratories). Sections were then incubated with rabbit anti–GFP polyclonal antibody (Ab 6556; Novus Biologicals), followed by biotinylated donkey anti–rabbit F(ab′)₂ (Jackson ImmunoResearch Laboratories), streptavidin-peroxidase, and FITC tyramide from the TSATM-fluorescein kit according to the manufacturer's instructions (PerkinElmer). Sections were quenched and blocked as described above and incubated with biotinylated anti-TCRβ antibody (H57; BD Biosciences), followed by streptavidin-Cy5 (Caltag). Biotin was blocked as described above, and sections were incubated sequentially with biotinylated anti–IFN-γ antibody (XMG1.2; BD Biosciences), streptavidin-peroxidase, and biotinyl-tyramide. Deposited biotin was detected by streptavidin-Cy3 (Caltag). Nuclei were counterstained for 5 min with a 10 μg/ml solution of 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Boehringer) in PBS. Slides were mounted in Vectashield (Vector Laboratories). Digital images in the DAPI, FITC, Cy3, and Cy5 channels were collected using a deconvolution fluorescence microscope equipped with Slidebook software (Intelligent Imaging Innovations). Images were converted to RGB, colored, and overlaid using Adobe Photoshop 5.5 software. IFN-γ levels and GFP levels were set against isotype control (biotinylated rat IgG1) and wild-type BALB/c tissue, respectively.