Anti sheep nl557

Animals were deeply anesthetized with an overdose of isoflurane and were perfused cardially with 20 mL of PBS. Brains were then excised and incubated in 4% paraformaldehyde (PFA) overnight and transferred to 30% sucrose solution for 48 h at 4 °C. The tissues were frozen and stored at −80 °C. Immunohistochemical staining was performed on free-floating 50-μm sections cut in a cryostat. For IbaI, CD11b, and CD39 staining, sections were washed with PBS and then incubated with PBS with 0.05% triton. The sections were then incubated in primary antibody diluting buffer (Golden Bridge International, Mukilteo, WA, USA) and then transferred for overnight incubation with IbaI antibody (WAKO, Osaka, Japan), CD39 (R&D, Minneapolis, MN, USA), and CD11b (Serotec, Raleigh, NC, USA) at 4 °C. The sections were then washed with PBS and incubated for 1 h with AlexaFluor 488 or 546 goat anti-rabbit (Invitrogen, Carlsbad, CA, USA) for IbaI staining and anti-sheep NL557 (R&D) for CD39 staining. Where indicated, slices were counterstained with TO-PRO-3 iodide (642/661) (Invitrogen).

Confocal microscopy was used to assess expression of Sema4C and its co-localization with molecules on murine and human B-cells. Cells cultured in eight-well slide chambers were fixed with 4% formaldehyde. They were blocked with Protein Block (Dako, Burlington, ON, Canada) for 45 min, and stained with primary antibodies for 1 h, followed by incubation for 1 h with the secondary antibody at room temperature. Nuclei were stained with Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) for 15 min at room temperature, and photomicrographs were taken with a Zeiss LSM780 Laser Scanning Confocal microscope.
Spleens from WT and Sema4C^−/− mice were snap-frozen, cryosectioned, and stained for histomorphometric measurement with ImageJ software (https://imagej.nih.gov/ij/).
Antibodies used included anti-mouse Sema4C (R&D Systems), anti-human Sema4C (R&D systems), anti-human Plexin-B2, anti-mouse Plexin-B2 (eBioscience), anti-human IgG (Acris), anti-sheep NL557, anti-sheep NL493 (R&D Systems), anti-mouse Alexa555 (Invitrogen), Phalloidin-Alexa Fluor 488 (Cytoskeleton), Cholera Toxin B-Alexa Fluor 488 (Sigma), and anti-actin (Millipore).