Figure 2.

Intravenous cytochrome c protein (cyt c) administration induces rapid contact between activated antigen-specific T cells and B cells. Splenic cryosections from 5C.C7 αβ T-cell receptor (TCR) transgenic mice, injected 1 hr previously with phosphate-buffered saline (PBS) (a) or 70 µg cyt c in PBS (panels b and c), were co-stained with phycoerythrin (PE)-conjugated anti-B220 and horseradish peroxidase (HRP)-conjugated MCC 88-103/I-E^k tetramer (panels a and b) or with PE-conjugated anti-B220 and biotinylated anti-CD69 (c), followed by avidin HRP, and developed using the tyramine signal amplification (TSA)-direct green signal amplification kit. B cells are red and activated T cells are green. Yellow interfaces between green and red cells indicate close T–B contact. Images are representative of tissue sections from 20 PBS-injected and four cyt c-injected mice at 1 hr. A 20× objective with confocal microscopy (panels a and b) or a 60× objective with standard fluorescence microscopy (panel c) were used.