In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Summary

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Abstract

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37°C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

Protocol

Adoptive Transfer

Inject neonates with 50-100ul of bone marrow using an insulin syringe 2-4 times during the first 3-7 days of life, with a final donor-to-host ratio of 1:1. Aim for an injection site below the sternum and rib cage, but above the gut and stomach. Insert the needle far enough to penetrate the peritoneum, careful not to puncture the diaphragm. It is not uncommon for some of the bone marrow to be lost during the injection. You can reduce the loss by loosening your grip on the pup as you inject so as to allow the skin to expand and, then, slowly removing the needle.

Dissection of Thymus

Discussion

Two-photon imaging in the thymus enables us to examine in living, intact organs the interactions and migrations that underlie selection events within the thymus.

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