Anti b220 efluor 450

Three weeks after the beginning of the treatment the spleens were harvested, mashed through a 70 µm nylon mesh (Corning) into single-cell suspension and the red blood cells were lysed using ACK solution (Thermo Fisher Scientific). Cells were stained with live/dead stain (anti-B220 eFluor 450 (eBioscience) and anti-CD138 PE (Becton Dickinson) antibodies, and analyzed by flow cytometry on LSR II Fortessa (Becton Dickinson) instrument. MM cells gating strategy is presented in Supplementary Fig. 1.

Spleens were snap-frozen in O.C.T. medium (Tissue-Tek; Sakura, Alphen aan den Rijn, The Netherlands). Cryosections (10 μm) were fixed in ice cold acetone for 10 minutes, air-dried, and blocked in 1% BSA plus 20% FBS in PBS before staining with Abs at room temperature. Cryosections were incubated with rat anti-mouse Siglec-1 (clone SER-4; provided by Dr. J. den Haan, VUMC, Amsterdam, The Netherlands) followed by Alexa555-conjugated goat anti-rat IgG (Invitrogen), anti-SIGN-R1 Alexa647 (clone 22D1; Dr. J. den Haan) and anti-B220 eFluor450 (eBioscience). Images were acquired using a Zeiss LSM 710 confocal laser scanning microscope with Zen software (Carl Zeiss Microimaging, Jena, Germany).