Alexa fluor647 conjugated anti mouse cd45r

The spleen of immunized mice was separated on day 7 after the final immunization and embedded in optimal cutting temperature (OCT) compound (SAKURA). The tissues were frozen with liquid nitrogen before sectioning (7 µm) on a cryostat. After being fixed in cold acetone and blocked with 5% FBS in PBS at RT for 1 h, the sections were incubated with Biotinylated PNA (VECTOR, 1:100) overnight at 4°C. DyLight 488 Streptavidin (BioLegend, 1:100) was used as the secondary antibody at RT for 1 h followed with Alexa Fluor647-conjugated anti-mouse CD45R (BioLegend, 1:150) at RT for 1 h. After staining, the sections were scanned under a Pannoramic SCAN instrument (3DHISTECH, Hungary).

The spleens from BALB/c mice were separated one week after the final immunization and embedded in Optimal Cutting Temperature (OCT) Compound (SAKURA, USA). The tissues were frozen at -80°C before sectioning (8 μm) on a Cryostat (Leica, Germany). After being fixed in cold acetone and blocked with 1% BSA in PBS at room temperature for 1 h, the sections were incubated with Biotinylated Peanut Agglutinin (PNA, 1 : 100 dilution, VECTOR, USA) at 4°C overnight. DyLight™ 488 Streptavidin (1 : 100 dilution, BioLegend, USA) was used as the secondary antibody at room temperature for 1 h. At last, Alexa Fluor® 647-conjugated anti-mouse CD45R (1 : 150 dilution, Clone RA3-6B2, BioLegend, USA) was incubated at room temperature for 1 h. The sections were scanned under a Pannoramic SCAN instrument (3DHISTECH, Hungary). For quantification the area of GCs, spleen sections of three mice from each group were analyzed by ImageJ software.