Gl7 af488

For detection of antigen-specific GC B cells, freshly isolated LN cell suspensions from individual mice were stained with Aqua Viability Dye (Thermo) and Fc blocked with an anti-CD16/32 antibody (clone 93; BioLegend). Cells were then surface stained with the relevant probes (APC or PE labelled stem, HEL, OVA or gp120 proteins for baiting antigen-specific GC B cells) [10 (link)] and the following antibodies: CD45 APC-Cy7 (30-F11; BD), CD3 BV785 (145-2C11; BioLegend), F4/80 BV785 (BM8; BioLegend), Streptavidin BV785 (BD), B220 BUV737 (RA3-6B2; BD), IgD BUV395 (11-26c.2a; BD), CD38 PE-Cy7 (90; BioLegend), GL7 AF488 (GL7; BioLegend). For detection of Tfh cells ex vivo, freshly isolated LN cell suspensions from individual mice were stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD Biosciences), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD Biosciences), CXCR5 BV421 (L138D7; BioLegend), PD-1 BV786 (29F.1A12; BioLegend). For Ag-specific Tfh identification [23 (link)], cells were cultured as described above and then stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD), CD25 BB515 (PC61; BD), OX40 PeCy7 (OX-86; BioLegend). All samples were acquired on a BD LSR Fortessa using BD FACS Diva and data was analyzed in FlowJo v10.

Kidneys and spleens were embedded in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA) and rapidly frozen in a dry ice and 2-methylbutane freezing bath. The frozen OCT-embedded samples were cryosectioned, and unstained slides were stored at -80°C. Subsequently, these frozen slides were allowed to reach room temperature and dry for 30 minutes before fixation in -20°C cold acetone for 10 minutes. After a wash in PBS, the slides were blocked with PBS containing 1% BSA and anti-mouse CD16/32 (1:100, BioLegend) for 40 minutes at room temperature. Following the blocking step, the slides were incubated with a mixture of fluorochrome-conjugated antibodies for 2 hours at room temperature in a humid box. Finally, the slides were mounted using Prolong Gold containing DAPI (Life Technologies). The immunohistochemical analysis utilized the following anti-mouse antibodies: complement C3-FITC (1:200, Cedarlane, Cat# 1850362A); IgG2a-PE (1:200, BioLegend, Cat# 407107); CD3-APC (1:40, BioLegend, Cat# 100235); GL7-AF488 (1:80, BioLegend, Cat# 144606); and IgD-PE (1:100, BioLegend, Cat# 405705). Slides were examined and imaged using a Zeiss LSM880 confocal microscope. Image processing and quantification of the fluorescent intensity was performed with ImageJ and ZEN 2.1 Lite software equipped with a 20× objective.