Cd4 cf 594 rm4 5

The following Abs/reagents were used for confocal immunofluorescent analysis: biotinylated anti-mouse IgD (11-26/SBA-1, SouthernBiotech), CD4-CF594 (RM4-5, BD biosciences), and streptavidin-Alexa Fluor 647 (Life Technologies) or BCL6-A647(clone K112-91, BD-Pharmingen) and IgM^a-PE (clone DS-1, BD-Pharmingen). Brachial lymph nodes (BLNs) were harvested from recipient mice, fixed for 1 h in 1% paraformaldehyde in PBS on ice and washed with PBS. They were then blocked overnight in 30% sucrose, 0.1% NaN in PBS, embedded in Tissue- Tek optimum cutting temperature compound, snap-frozen in dry ice and ethanol, and stored at −70 °C. Thirty micron cryostat sections were cut from the tissue blocks, affixed to Superfrost Plus microscope slides (Fisher), and stained first with biotinylated anti-IgD Abs and then with anti-CD4 Abs and streptavidin as previously described [22 (link)]. Alternatively they were stained with anti-Bcl6 and anti-IgM^a Abs. Confocal analysis of the sections was performed using Leica SP5 with argon and helium-neon lasers, 2-channel Leica SP spectral fluorescent PMT detector, and a 20× oil-immersion objective with a numerical aperture of 0.7. Images were processed using Imaris (Bitplane).