Inguinal lymph nodes were harvested, immersed in Optimum Cutting Temperature (OCT) compound (VWR Chemicals), and immediately frozen on dry ice. Thirty-five-micrometer sections were cut using a cryostat (Leica), mounted on Superfrost Ultra Plus slides (Thermo Scientific Gerhard Menzel), fixed in acetone for 10 min at −20°C, air-dried for 30 min, and stored at −20°C.
Prior to staining, non-specific Ab binding was blocked using a solution of 1% v/v normal mouse serum, 2% w/v BSA (Sigma-Aldrich), and 0.3% v/v Triton X-100 (Sigma-Aldrich). Primary Abs and secondary reagent were used as detailed inSupplemental Table I . After final washing, Fluoromount G mounting medium (eBioscience) was applied, followed by a coverslip.
Slides were visualized using a Zeiss LSM 710 confocal microscope. Images were acquired using Zen software (Zeiss). ImageJ (National Institutes of Health) was used for image processing. Images were analyzed in a manner allowing comparison of both overall GC area and proportion of the B cell area occupied by GCs: GC and B cell areas were identified using GL7 and B220 staining, respectively, manually defined, and their areas were calculated (44 (link)).
Prior to staining, non-specific Ab binding was blocked using a solution of 1% v/v normal mouse serum, 2% w/v BSA (Sigma-Aldrich), and 0.3% v/v Triton X-100 (Sigma-Aldrich). Primary Abs and secondary reagent were used as detailed in
Slides were visualized using a Zeiss LSM 710 confocal microscope. Images were acquired using Zen software (Zeiss). ImageJ (National Institutes of Health) was used for image processing. Images were analyzed in a manner allowing comparison of both overall GC area and proportion of the B cell area occupied by GCs: GC and B cell areas were identified using GL7 and B220 staining, respectively, manually defined, and their areas were calculated (44 (link)).
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