Mice were anesthetized with isofluorane and maintained at a core temperature of 37°C. The peritoneum was opened minimally to access omentum. A small portion of visceral adipose tissue was resected to assist in immobilizing the omentum via a soft-tissue vacuum apparatus (VueBio). No greater than 40 kPa of pressure was applied to steady the omentum for imaging. Image acquisition was performed on a Leica SP8 multiphoton confocal with a 20× water immersion objective (1.0 NA) with a resonant scanner (8,000 kHz) and four external HyD detectors. The excitation wavelength of Chameleon Vision II Ti:Sapphire laser was tuned for optimal detection of CellTrace dye–labeled CD8+ T cells in each experiment, typically 880 nm. Images collected x-, y-, and z-plane data over time with a step size of 2 μm and a z-thickness that allowed for a complete z-series every 22 s. This was carried out for ∼30 min for each region imaged. The resulting images were segmented in Imaris (v9.7.2; Bitplane) with spot-specific position data exported and analyzed in R with the CellTrackR package (Wortel et al., 2021 (link)).