Apc labeled anti b220

Spleens were harvested from the mice after NP-Ficoll immunization at indicated times, snap-frozen in liquid nitrogen and frozen in Tissue-Tek OCT compound (Sakura) at −80°C. For immuno-fluorescence of IDO1 5μm frozen splenic sections were immediately fixed in −20°C methanol. The sections were blocked with PBS containing 1% nonfat milk (Sigma) and stained with 4μg/mL polyclonal rabbit anti-mouse IDO1 (a gift from Dr. David Munn) in PBS containing 1% nonfat milk. After extensive washing with TBS + 0.05% tween 20 (ACROS Organics), the sections were stained with a 1:400 dilution of CY3-labeled goat anti-rabbit IgG (Jackson Immunoresearch), APC-labeled anti-B220 (Ebioscience), or FITC-labeled anti-CD138 (BD Pharmingen). To examine antibody response to NP-Ficoll immunization, sections were incubated with anti-IgM (BioLegend) and anti-IgG3 (Southern Biotech) in blocking buffer for 60 minutes at room temperature in the dark. For stimulated and unstimulated purified B cells, cytospins were prepared and fixed with −20°C methanol. Cells were made permeable using Perm Buffer III (BD Biosciences) and stained for IDO1 as explained above and anti-B220 (eBioscience). Sections and cytospins were mounted with Prolong Gold anti-fade with DAPI (Invitrogen). Fluorescent images were captured using a Zeiss LSM 510 Meta confocal microscope equipped with 405-, 488-, 561-, and 633-nm lasers.