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.
Authors
- Miu Shing Hung (ORCID: https://orcid.org/0009-0004-0412-4374)
- Alessandro Ciccarelli (ORCID: https://orcid.org/0000-0002-7612-8161)
- Dinis Pedro Calado (ORCID: https://orcid.org/0000-0001-8239-7184)
- Anita Grigoriadis (ORCID: https://orcid.org/0000-0003-3434-201X)
- Victoire Boulat
- Lingling Zhang (ORCID: https://orcid.org/0000-0001-7423-3761)
Institutions
- King's College - North Carolina (US)
- King's College London (GB)
- The Francis Crick Institute (GB)
Publication Details
- Journal
- STAR Protocols
- Published
- 2025-09-01
- DOI
- https://doi.org/10.1016/j.xpro.2025.104059
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Wellcome Trust
- Fonds der Chemischen Industrie
- Cancer Research UK
- Medical Research Council
- Biotechnology and Biological Sciences Research Council