1.4 na plan apochromat oil immersion objective

Fluorescence detection was carried out using a Carl Zeiss LSM 880 microscope equipped with a spectral detector and a 63× NA 1.4 plan apochromat oil immersion objective (Zeiss, Oberkochen, Germany). Enhanced green fluorescent protein and Alexa 488 fluorophores were excited by a 488 nm argon laser line and its emission was detected in a range of 490–543 nm. The Helium laser line of 546 nm and a detection range of 549–626 nm were used to detect Cy3 fluorophore emission. The mTurq2 and seyfp2 fluorescent proteins were excited by 458 and 514 nm argon laser lines, respectively. A detection range of 456–510 nm for mTurq2 and 516–561 nm for seyfp2 were used. Cross-excitation of all these fluorophores was negligible and cross-emission was eradicated by using the multi-track mode. In these experiments, although the fluorescent proteins fused to Tax or DLG1 changed and had different spectral properties among the experiments, in all cases the Tax expression was shown in green and the DLG1 expression in red. The same strategy was followed for the organelle markers, whose expression was shown in blue. The co-localization among proteins was assessed by the superposition of images obtained in the same confocal sections.

TILs were conjugated with sAg-pulsed CP50-EBV cells previously stained with 5-(and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; 1 μM; Molecular Probes) at ratio 1:1 by 1 min centrifugation. After 25 min of coculture on slides coated with poly-D-lysine (50 μg ml⁻¹; Sigma-Aldrich), cells were fixed in 3% PFA, permeabilized with 0.1% saponin in PBS, 3% bovine serum albumin (BSA) and HEPES, and stained with AlexaFluor 488 phalloidin (Molecular Probes) or one of the following antibodies: anti-CD11a (clone EP1285Y, Abcam), anti-α-tubulin (clone DM1A, Sigma-Aldrich). Staining for γ-tubulin (a microtubule nucleator used to detect centrosome position) detects the position of centrosome in cells more precisely than staining for α-tubulin (to detect microtubule organization). Nevertheless it has been shown that in TILs MTOC positioning as indicated by α-tubulin staining co-localizes with centrosome staining56 (link). Primary antibodies were followed by appropriate fluorochrome-coupled anti-species isotype specific Ab (Molecular Probes). The samples were mounted in ProLong Gold (Molecular Probes). Random acquisition of T-cell-target conjugates was performed using a Zeiss LSM 510 confocal microscope with a × 63 NA1.4 Plan-Apochromat oil immersion objective (Carl Zeiss–Oberkochen, Germany). Images were processed using software ImageJ (National Institute of Health, USA).

Fixed sample images were acquired using an LSM 700 laser scanning confocal microscope with a 63× 1.4 NA Plan-Apochromat oil-immersion objective (Carl Zeiss). Confocal stacks were collected with a z step of 0.3 μm over 7–10 μm with pixel size of 100 nm (x–y image size 51 × 51 μm). Acquisition parameters were established using the brightest control specimen such that just a few pixels reached saturation in order to achieve the greatest dynamic range in our experiments. For quantitative measurements such as particle volume or fluorescence intensity, parameters including gain, laser power, scan speed, dwell time, resolution, and zoom, were kept consistent between comparisons.