Protocol for rapid 5-plex 3D imaging and single-cell analysis of immune responses in whole murine lymph nodes

Lymph nodes orchestrate adaptive immune responses, with germinal centers enabling affinity maturation and plasma cell formation. Here, we present a protocol for rapid, high-resolution, multicolor 3D imaging of whole immunized mouse lymph nodes. We describe steps for immunization, lymph node harvesting, fixation, permeabilization, staining, and clearing. We cover image acquisition using a light sheet fluorescence microscope and analysis using the Imaris software. This protocol allows the quantification of germinal center B cells, plasma cells, and follicular T cells at single-cell resolution.