Cd21 35 clone ebio4e3

For confocal microscopy of frozen spleen sections, spleens were removed at the indicated day post-infection, fixed in periodate-lysine-paraformaldehyde fixative for 48 h, and then moved to 30% sucrose/PBS solution for 24 h. Tissues were embedded in optimal-cutting-temperature medium (Electron Microscopy Sciences) and frozen in dry-ice-cooled isopentane. 16-µm sections were cut on a Leica cryostat (Leica Microsystems, Buffalo Grove, IL). Sections were blocked with 5% goat, donkey, bovine, rat, or rabbit serum and then stained with a combination of the following Abs: ERTR7 (Abcam, Boston, MA), B220 (clone RA3-6B2, Biolegend), GL7 (clone GL-7, Thermo Fisher Scientific), and CD21/35 (clone eBio4E3, eBioscience, San Diego, CA). Sections were incubated with secondary antibodies as needed and for controls, and images were acquired on a Leica SP8 or Stellaris Confocal microscope. To increase staining intensity of MHV68, spleens were stained with anti-GFP Ab (EPR14104, Abcam) and goat anti-chicken Alexa Fluor 488 (ThermoFisher Scientific, Waltham, MA). Images were processed and analyzed using Imaris software 8.0 (Oxford Instruments). Where indicated, the spots function of Imaris was used to identify and create spots for MHV68+ cells. Spots were masked on TdTomato expression inside the spot to reveal MHV68+ tdTomato+ cells, referred to as “gated.”