Infrared blanket

Mice were anesthetized in 1.5% isoflurane USP (Baxter) through a rodent nose cone followed by surgery to expose the right popliteal lymph node. Warm PBS was added to maintain lymph node moisture. Mouse body temperature was maintained with an infrared blanket (Braintree Scientific) throughout the imaging process after which they were euthanized through cervical dislocation under anesthesia. Mice were imaged on a glass stage with a two-photon laser-scanning microscopy setup with a Leica SP8 inverted confocal microscope with dual multi-photon lasers, Mai Tai and InSight DS (Spectra-Physics), L 25.0 water-immersion objective, 0.95 NA (Leica Microsystems), and a 37  °C incubation chamber (NIH, Division of Scientific Equipment and Instrumentation Services). The Mai Tai laser was tuned to 890  nm to excite e450 and FlAsH; the InSight DS laser was tuned to 1150  nm to excite DsRed/ PKH-26 /ReAsH and e670. For time-lapse imaging, a z stack consisting of 10–12 single planes (5  μm each over a total tissue depth of 50–60  μm) was acquired at 15 s intervals for a total period of 1–4 hr.

Mice were anesthetized in 1.5% isoflurane USP (Baxter) through a rodent nose cone followed by surgery to expose the right popliteal lymph node. Warm PBS was added to maintain lymph node moisture. Mouse body temperature was maintained with an infrared blanket (Braintree Scientific) throughout the imaging process after which they euthanized through cervical dislocation under anesthesia. Mice were imaged on a glass stage with a two-photon laser-scanning microscopy setup with a Leica SP8 inverted confocal microscope with dual multi-photon lasers, Mai Tai and InSight DS (Spectra-Physics), L 25.0 water-immersion objective, 0.95 NA (Leica Microsystems), and a 37°C incubation chamber (NIH, Division of Scientific Equipment and Instrumentation Services). The Mai Tai laser was tuned to 890 nm to excite e450and FlAsH; the InSight DS laser was tuned to 1,150 nm to excite DsRed/ PKH-26/ ReAsH and e670. For time-lapse imaging, a z stack consisting of 10–12 single planes (5 μm each over a total tissue depth of 50–60 μm) was acquired at 15 second intervals for a total period of 1–4 h.