Anti b220 alexa488

To analyze the formation of tertiary lymphoid follicles in the lungs following LVS infection, tissues were fixed in PLP buffer (2% paraformaldehyde, 0.05 M phosphate buffer containing 0.1 M l-lysine, and 2 mg/ml NaIO₄) overnight. Tissues were then dehydrated in 30% sucrose and subsequently embedded in OCT media. Twenty micron frozen sections were preincubated with Fc-block (anti-mouse CD16/32 Ab; Biolegend) diluted in PBS containing 2% goat serum and fetal bovine serum (FBS). After incubation for 1 hour at room temperature, sections were washed with PBS and stained with the following antibodies diluted in PBS containing 2% goat serum and FBS for 1 hour at room temperature: anti-B220-Alexa-488 (Biolegend), anti-CD3-allophycocyanin, anti-CD11c-phycoerythrin (Biolegend) and Fc-block. Sections were washed with PBS and mounted using Immu-Mount (Thermo Fisher Scientific). Images were acquired using a Zeiss 780 confocal microscope (Carl Zeiss). The imaging data were processed and analyzed using the Imaris software (Bitplane; Oxford Instruments).