B220 af647

Spleens were fixed in 4% methanol-free formaldehyde (Sigma, Cat. 28906) in PBS at 4 °C for 3.5 h followed by overnight incubation in 30% sucrose in PBS and freezing in OCT (Tissue-Tec, Cat. 4583). Spleens were cut into 10 µM sections using a Leica CM1850 cryostat and the tissue was transferred onto microscope slides. The tissue was blocked with PBS + 5% FCS + 2% rat serum + anti-FcR (clone 2.4G2, in-house hybridoma) for 1 h followed by staining with antibodies to Bcl6-CF568 (clone 7D1-10, in-house purified and conjugated to CF568 (Biotium, Cat. 92215); 1 in 200 dilution), CD35-biotin (BD Biosciences, Cat. 553816; 1 in 200 dilution), CD45.1-eFluor450 (clone A20, eBioscience, Cat. 48-0453-82; 1 in 200 dilution), CD45.2-FITC (clone 104, BD Biosciences, Cat. 553772; 1 in 200 dilution), and B220-AF647 (clone RA3-6B2, in-house purified and conjugated to Alexa fluor 647; 1 in 200 dilution) in a humidified chamber at 4 °C overnight. Slides were washed and stained with Streptavidin-Pacific orange (Thermo Fischer, Cat. S32365) at room temperature for 1 h. After washing twice for 1 h with PBS, slides were mounted using DAKO fluorescent mounting medium (DAKO, Cat. S3023) and Menzel X1000 Coverslip #1.5. Images were acquired on a Nikon AR1 confocal microscope with a CFI Plan Fluor ×20 MI lens using glycerol as immersion medium.

Peyer’s patches (PP) were isolated from the small intestine and cells were extracted as previously described^{16 (link)}. Briefly, PP were finely cut and incubated for 40 min at room temperature with collagenase/DNase. Cells were then filtered through a 70 µm cell strainer and pelleted by centrifuging at 300×g, 4 °C during 5 min and then resuspended in FACS buffer (PBS, 2% FCS, 5 mM EDTA) and counted before antibody staining. For flow cytometry, cells were first incubated on ice in FACS buffer containing an anti-CD16/CD32 antibody to block the Fc receptor for 10 min and then incubated 30 min on ice in the dark with antibodies against the following surface markers : CD45-PE-eF610 (eBiosciences, clone 30-F11), CD19-PerCp-Cy5.5 (Biolegend, clone 6D5), CD3-PeCy7 (eBiosciences, clone 145-2C11), B220-AF647 (BD Biosciences, clone RA3-6B2), IgD-BV421 (BD Biosciences, clone 11-26c.2a), IgM-FITC (BD Bioscience, clone II/41) and IgA-PE (eBiosciences, clone mA-6E1). Cell viability was evaluated using Fixable Viability Dye eFluor 506 (eBiosciences). Cell acquisition was performed using a CytoFlex (Beckman Coulter) and data were analyzed with the CytoExpert software (Beckman Coulter).