Protocol for genetic manipulation of cervical lymph node-innervating nociceptive neurons in mice via retrograde viral tracing

This protocol details the surgical procedure for genetically manipulating cervical lymph node (LN)-innervating nociceptive neurons in mice. We describe steps for exposing cervical LNs, microinjecting retrograde adeno-associated viruses (AAVs), and verifying viral transduction in the trigeminal ganglia. This approach enables specific labeling, chemogenetic inhibition, or gene knockout in LN-innervating neurons to study neuroimmune crosstalk. For complete details on the use and execution of this protocol, please refer to Zhang et al. 1 • Instructions for microsurgical exposure of mouse cervical lymph nodes • Steps for precise retrograde viral microinjection into cervical nodes • Guidance on verifying viral expression in trigeminal ganglia neurons Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. This protocol details the surgical procedure for genetically manipulating cervical lymph node (LN)-innervating nociceptive neurons in mice. We describe steps for exposing cervical LNs, microinjecting retrograde adeno-associated viruses (AAVs), and verifying viral transduction in the trigeminal ganglia. This approach enables specific labeling, chemogenetic inhibition, or gene knockout in LN-innervating neurons to study neuroimmune crosstalk.