Donkey anti rat igg cy5

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).