Anti mouse cd21 cd35 alexa fluor 594

For observing the germinal center structure in the spleen, part of the spleen from the immunized or infected mice was fixed in 4% Paraformaldehyde at 4°C overnight, then the spleens were dehydrated in 20% sucrose for at least 36 h. Then the spleens were embedded in OCT and sectioned at 5 μm thickness. The slides were air-dried at room temperature before being fixed in cold acetone at −20 °C for 10 min. The fixed slides were washed with PBS for twice and blocked with Blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature. Biotinylated Peanut Agglutinin (PNA, Vector laboratories) was stained on the sections at 4°C overnight. The slides were washed with PBST 3 times, then the Alexa Fluor^™ 488 Conjugated Streptavidin, anti-mouse CD21/CD35 (Alexa Fluor^® 594, Biolegend), and anti-mouse IgD (Alexa Fluor^® 647 anti-mouse IgD, Biolegend) was applied onto the section for 2 h at room temperature. After washing with PBST for 3 times, the slides were counterstained with 4’, 6-diaminodino-2-phenylindole (DAPI) and mounted. The stained slides were reviewed, and representative images were acquired on Olympus DP80 digital microscope.