Gl 7 biotin

Spleens were fixed in buffered formol for 12 h and paraffin-embedded. Splenic tissue sections (5 mm) were hematoxylin-eosin stained using standard procedures. Spleens were harvested and immediately embedded in Tissue-Tek O.C.T. (Sakura Finetek, Japan) and snap frozen. Slices measuring 8 μm thick were fixed in acetone and blocked with 3% BSA plus Fc Block (1:100) (BD Bioscience) for 1 h at 24°C. Slides were then stained with anti-CD4-Alexa 700 (RM4-5) (eBioscience), CD19-APC (ID3), GL-7-Biotin (GL7) and Streptavidin-FITC (BD Biosciences) for 2 h at 24°C. After washing, slides were stained with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes) and mounted with Vectashield mounting medium (Vector, USA). Sections were analyzed by confocal microscopy (Zeiss LSM 780, Germany).

Single immune cell suspensions were prepared from spleen or peritoneal lavage after red blood cell lysis. Cells were counted on a BD Fortessa Flow cytometer using ‘Fluoresbrite ^TM Calibration Grade 6.0 micron YG microspheres (Polysciences). Cells were incubated with anti-CD16/CD32 (Fc block, clone 2.4G2; BD Bioscience) plus 2% normal mouse and normal rat serum and stained with various combinations of the following Abs in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) for 30 min at 4°C. Murine reactive antibodies, including B220-Pacific Blue, CD3-PerCPCy5.5, CD4-Pacific Blue, CD5-PerCPCy5.5, CD8-APC Cy, CD11b APC Cy7, CD21 FITC, CD23 PE, CD38-FITC, CD95 PE Cy7, CD138-PE, IgD APC Cy7, IgM FITC, GL-7 biotin, Streptavidin BV500, Ki67-APC, Ly6G PE Cy7, and Ly6C PerCP Cy5.5 were all purchased from BD Bioscience.