Moma 1

Spleens and LN from naïve mice were harvested and embedded in Tissue-Tek OCT (Sakura Finetek), then rapidly frozen by immersion in liquid nitrogen-cooled 2-methyl butane, and kept at -80°C overnight. Cryostat sections (10–12 μm) were cut at -20°C, mounted on glass slides, air-dried for 2–3 hours, fixed for 10–15 minutes in cold acetone, air-dried again for 30 minutes, and stored at -80°C. Slides were warmed to room temperature and stained in 1x TBS with 5% BSA and 0.1% Tween 20. Sections were stained with antibodies to SIGNR1 (22D1), secondary labeled with mouse anti-hamster Alexa Fluor 488 (1:100 dilution). CD169 was visualized with the clone MOMA-1 (Abcam), biotinylated and secondary labeled with streptavidin-PE (1:200 dilution). Microscope and software used to analyze microscopy were from Leica Microsystems (Buffalo Grove, IL).
To visualize infection of systemic macrophages with VACV we infected mice with 10,000 pfu VACV-GFP (strain Western Reserve, with no gene deletions). Six hours later spleens were harvested, flash frozen, sectioned (10 μm) and fixed in 2% paraformaldehyde in PBS (pH 7.4). Sections were then stained with antibodies to CD169 and SIGN-R1 as above.

The following staining reagents were used: anti-CD3 (145-2C11), anti-CD4 (H129.19), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-CD45R (RA3-6B2), anti-PD-1 (29F.1A12), anti-PD-L1 (10F.9G2), anti-ICOSL (HK5.3), anti-CD40 (HM40-3), anti-CD40L (MR1), anti-CXCR4 (L276F12), and anti-IL-4 (11b11) were from Biolegend (San Diego, CA, USA); anti-CD19 (eBio1D3), anti-ICOS (7E.17G9), anti-IL-21 (mhalx21), anti-CD69 (H1.2F3), and anti-GL7 (GL-7) were from eBiosciences (San Diego, CA, USA); anti-Bcl-6 (K112-91), anti-CXCR5 (2G8), and anti-Fas (Jo2) were from BD Biosciences (Franklin Lakes, NJ, USA). PNA (B-1075; Vector Laboratories, Burlington, ON, Canada) was used for staining germinal center B cells and MOMA-1 (Abcam, Cambridge, UK) for staining MZ macrophages.