Tc 324b temperature controller

Two-photon imaging of thymic slices has been previously described (Melichar et al., 2013 (link); Dzhagalov et al., 2012 (link); Ross et al., 2014 (link); Dzhagalov et al., 2013 (link); Au-Yeung et al., 2014 (link)). All imaging was performed 3–6 hr after the addition of thymocytes to thymic slices. Thymic slices were glued onto glass cover slips and fixed to the bottom of a petri dish being perfused at a rate of 1 ml/min with 37°C oxygenated, phenol red–free DMEM during imaging using an MPII Mini-Peristaltic Pump (Harvard Apparatus) and a 35 mm Quick Exchange Platform (QE-1) in conjunction with a TC-324B temperature controller (Warner Instruments). Samples were imaged in the cortex of the thymus, determined by proximity to the thymic capsule. Images were collected using a Zeiss LSM 7 MP upright, two-photon microscope with a 20×/1.0 immersive Zeiss objective and a Coherent Chameleon laser tuned to 720 nm for imaging Indo-1. Ratiometric Ca²⁺ signals were collected with 440-nm-long pass dichroic mirror and 400/45 and 480/50 bandpass filters. Ca²⁺ hi signals (~401 nm) were collected using a bialkali PMT; Ca²⁺ lo signals (~475 nm) were collected using a GaASP PMT. Image areas of up to 212 × 212 μm to a depth of up to 55 μm were acquired every 10 s for 15 or 20 min with 3 μm z steps starting from beneath the cut surface, using Zen 2010 software from Zeiss.