Anti baff r

For histology and immunostaining, 8 µm sections were cut using a microtome (SLEE medial, Mainz, Germany). Russell-Movat pentachrome (Abcam, Cambridge, UK) or Alizarin Red staining (Sigma Aldrich, St. Louis, MO, USA) were performed according to the manufacturer’s instructions. For immunohistochemistry analysis, after antigen retrieval with EDTA buffer (Sigma-Aldrich, St. Louis, MO, USA), slices were incubated overnight with an anti-BAFF-R or CD138 antibodies (Abcam, Cambridge, UK) diluted at 1:250 or 1:8,000, respectively. Slides were stained with HRP/DAB detection kit (Abcam, Cambridge, UK) and counterstained with hematoxylin to visualize cell nuclei. Images of entire aortic valve cusps were composed using NIS-Elements Microscope Imaging Software (Nikon, Tokyo, Japan) from a series of adjacent pictures taken with a 4 × objective taken with a Nikon Eclipse Ni microscope (Nikon, Tokyo, Japan). Immunostaining images were taken with a 20 × objective with a Nikon Eclipse Ni microscope (Nikon, Tokyo, Japan).

RIPA-soluble proteins (20 μg) from spleen or total aorta were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-AID, anti-BAFFR (Abcam, MA) and anti-β-actin antibodies (Santa Cruz Biotechnology, TX) overnight at 4 °C. Regarding the western blot images in Supplemental Fig. 1, the membrane was cut at 35 kDa, allowing for incubation of the upper part with anti-beta-actin and the lower part with anti-AID antibodies.
Membranes were washed three times with TBST and incubated with HRP-conjugated secondary antibody (Bio-Rad Laboratories, CA). Membranes were revealed by chemiluminescence with the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, CA) and quantified by densitometry using Quantity One software (Bio-Rad).