Preparation of mouse tumor slices for ex vivo two-photon microscopy

Immune cell behavior in tumors can be visualized in real time by imaging ex vivo tumor slices. Here, we present a protocol for preparing mouse tumor slices for ex vivo two-photon microscopy. We describe techniques for adoptive transfer of fluorescent T cells, generation of tumor slices by vibratome, time-lapse two-photon imaging, and quantitative analysis. This protocol provides a model system to study immune cell migration and interactions within solid tumor microenvironments without the technical challenges of intravital microscopy.