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The Petri dish was filled with sterile phosphate buffered saline (PBS) to act as an ultrasound conduction medium for imaging, thus avoiding the use of ultrasound gel.

The camera sensitivity was set to "high" for imaging through a closed cranial window, and "medium" for imaging through a thinned skull cranial window.

The standard extracellular medium for imaging and electrophysiological measurements consisted of (in mM) 138 NaCl, 5.6 KCl, 1.2 MgCl2.62.6 CaCl2, 5 HEPES, and 5 D-glucose at pH 7.4 (adjusted with NaOH).

For TCEP treatment at 4 °C, we used 10 mM TCEP for 15 min. At this point, cells can be returned to complete medium for imaging or further processing or fixed for fluorescence analysis.

To visualize NKT cells located in deeper regions of the spleen (MZ and PALS) we performed an alternative imaging approach by cutting open the spleen across the transverse axis and placing the tissue in warm oxygenated medium for imaging (as previously reported (Aoshi et al, 2008; Bajénoff et al, 2008)).

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Cells were transfected with siRNA oligos or plasmid vectors for 48 or 24 hr, respectively, prior to imaging and were transferred to Leibovitz's L15 medium (Invitrogen) for imaging at 37°C.

Cells were transfected with siRNA oligos or plasmid vectors for 48 or 24 h, respectively, prior to imaging and transferred to Leibovitz's L15 medium (Invitrogen) for imaging at 37°C.

Cells were washed four times with PBS at RT for 5 min each, incubated with secondary fluorescent antibodies at 1 400 dilution in PBS with 1% BSA and 0.05% saponin for 60 min, re-washed with PBS, and then mounted with Fluoro-Gel anti-fade mediumng mEMSum (EMS) for imaging.

Intravenous administration of contrast medium is imperative for imaging polytrauma patients, and as there is usually no luxury of time, only post-enhanced imaging is performed so as not to miss any injury of the major mediastinal vessels and the heart; optimal opacification may be obtained with injection of 100 140 ml of iodinated contrast medium at a flow rate of 3 4 ml/s and a delay of 25 40 s.

By avoiding radiation exposure and administration of intravenous contrast medium, DWI is especially attractive for imaging children.

Cells (~ 3.5 × 106 DU145 or DU145-PSMA cells) were injected subcutaneously on the flank in 90 μl of RPMI 1640 medium, and animals were used for imaging or ex vivo biodistribution studies after 25 days when tumours reached ~ 5 mm in diameter, as determined using a Vernier caliper.

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