Anti b220 pe

The mice were deeply anesthetized and perfused transcardially with cold phosphate-buffered saline (PBS) followed by perfusion with 4% paraformaldehyde. The spinal cords and spleens were dissected carefully and cryo-protected in 30% sucrose. After embedded in optimal cutting temperature compound, the specimens were sectioned into 10-μm-thick sections using a cryostat microtome. For Tfh-like cells staining, sections were incubated in the dark with anti CD4-FITC (1:100; eBioscience), rabbit anti-mouse CXCR5 (1:500; Merck Millipore, Darmstadt, Germany), anti ICOS-PE (1:100; eBioscience), or anti PD-1-PE (1:100; BD Biosciences, San Jose, CA, USA) at 4°C overnight, followed by incubation with donkey anti-rabbit IgG-Cy5 (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 2 h at room temperature (RT). For GC and ELSs staining, sections were first incubated with rat anti-mouse CD35 (1:100; BD Biosciences) at 4°C overnight, followed by incubation in the dark with donkey anti-rat IgG-Cy5 (1:1000; Jackson ImmunoResearch Laboratories) for 2 h at RT. After CD35 staining, sections were blocked with 10% rats serum for 1 h at RT, and then incubated in the dark with anti CD4-FITC (1:100) and anti B220-PE (1:100; both from eBioscience) at 4°C overnight. Immuno-stained sections were visualized using confocal laser scanning microscopy (BX51, Olympus Corp., Tokyo, Japan).

Cell suspensions were incubated for 5 minutes with 10% rat serum (MP Biomedicals) and 0.2% BSA (Roche) in PBS, then stained for 30 minutes at 4°C with biotinylated lineage markers (Mac1, Gr1, CD4, CD8, B220, Ter119, IL7Rα, CD19, and CD3 with streptavidin PerCPcy5.5 as secondary), anti-Sca1-PE, anti-cKit-APC, anti-Gr1-FITC, anti-Mac1-APC, biotinylated anti-CD4 and anti-CD8 (with streptavidin APC as secondary), and/or anti-B220-PE (eBioscience and BD Biosciences). The analyzer used for flow cytometry was the BD LSR II and data was analyzed using CellQuest.

Single-cell suspensions were prepared from the spleen, bone marrow, and thymus. Red blood cells were lysed by homemade RBC lysis buffer, and remaining cells were incubated with antibodies in PBS for 30 min at 4 °C and washed with PBS twice before analyzed by BD FACSAria III. Antibodies used for staining include as follows: anti-B220-PE (eBiosciences, 12-0452-83), anti-TCRβ-APC (eBiosciences, 17-5961-82), anti-CD4-PE (eBiosciences, 12-0041-85), and anti-MHCII-APC (eBiosciences, 17-5321-81). FACS results were analyzed by FlowJo.

Cell suspensions were incubated for 5 minutes with 10% rat serum (MP Biomedicals) and 0.2% BSA (Roche) in PBS, then stained for 30 minutes at 4°C with biotinylated lineage markers (Mac1, Gr1, CD4, CD8, B220, Ter119, IL7Rα, CD19, and CD3 with streptavidin PerCPcy5.5 as secondary), anti-Sca1-PE, anti-cKit-APC, anti-CD34-FITC, anti-CD16/32-PEcy7, anti-Gr1-FITC, anti-Mac1-APC, biotinylated anti-CD4 and anti-CD8 (with streptavidin APC as secondary), anti-B220-PE, anti-CD71-PE, and/or anti-CD36-APC (eBioscience and BD Biosciences). The analyzer used for flow cytometry was the BD LSR II and data was analyzed using CellQuest.