Fitc anti mouse cd68

Mouse spleens were collected at 7 days post immunization, embedded in OCT medium and frozen at −80 °C before processing into 5 μm sections. Samples were fixed in cold 4% paraformaldehyde for 10 min followed by blocking in 5% FBS for 1 h at room temperature. For GC analysis, Alexa-Fluor 594 B220 (Biolegend 103254), Alexa-Fluor 488 anti-mouse IgG (Biolegend 405319), and Alexa-Fluor 647 anti-GL-7 (Biolegend 144605) or for macrophage depletion, Alexa-Fluor 594 B220, FITC anti-mouse CD68 (Biolegend 137005) /FITC anti-mouse CD11c (Biolegend 117305), and Alexa-Fluor 647 anti-mouse CD169 (Biolegend 142407) were incubated at 1:100 overnight at 4 °C. Samples were mounted on glass slides in ProLong Antifade reagent (Invitrogen). Images were acquired on a Zeiss LSM700 confocal microscope and on a Mirax Midi slide scanner system for whole organ imaging. GCs were defined as discrete areas of B220, GL-7, and IgG signal colocalization and counted manually in each spleen section. PBs were defined as IgG⁺ cells and quantified in two 1 mm² areas in each spleen section using ImageJ.

For determination of M2 macrophage expression, cells were labeled with FITC anti-mouse CD68 (BioLegend, San Diego, CA) and APC anti-mouse CD206 (BioLegend). After incubation with antibodies at 4°C for 30 min, cells were washed with PBS supplemented with 2% BSA, 4 mM EDTA, and 0.01% NaN3 and fixed in 4% paraformaldehyde. Data were collected on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software.