Af647 sav

Spleens from immunized mice were frozen in optimal cutting temperature (OCT) compound (Tissue-Tek cryomold and OCT gel compound, Sakura Finetek USA, Torrance CA) and cryosectioned in 5μm slices. Cryosections were fixed in cold acetone for 10 min at −20°C, washed with PBS, and then solubilized in 0.5% Tween-20 in PBS. After further washing in PBS, endogenous biotin and streptavidin binding sites were blocked according to manufacturer’s instructions (SP-2002, Vector Laboratories, Burlingame CA). For germinal center detection, sections were sequentially incubated with the following primary antibodies for 30 min at RT: CD3-FITC (17A2, eBioscience, San Diego CA, 1:100); CD45R/B220 (RA3–6B2, BD, Franklin Lakes NJ, 1:200); peanut agglutinin-biotin (B-1075, Vector Laboratories, Burlingame CA, 1:100). Slides were washed with 0.1% Tween-20 in PBS, and then incubated with secondary antibodies for 30 min at room temperature (AF555-goat anti-rat IgG (Invitrogen, Carlsbad CA, 1:500); AF647-sAv (Invitrogen, Carlsbad CA, 1:200)). After washing, slides were mounted with Vectashield Mounting Medium (H-1000, Vector Laboratories, Burlingame CA). Images were acquired on a Zeiss LSM 700 Confocal Microscope (magnification 200x) and pictures processed using Zen (Zeiss, Oberkochen Germany) and QuPath software.