4.7. Immunofluorescence and Confocal Laser Scanning Microscopy

To investigate the subcellular localization of Tax and ELL2, 1.5 × 10⁵ 293T cells/experiment were seeded on coverslips. After 24 h, cells were transfected with expression plasmids pEF1α-ELL-myc (1 µg) or pEF1alpha Tax (1 µg), supplemented with the respective empty control vector pEF-1α to 2 µg. After another 24 h, cells were fixed with 4% para-formaldehyde (PFA, 45 min, humid chamber, 20 °C), washed twice in PBSo (phosphate-buffered saline without Ca²⁺ and Mg²⁺), and permeabilized with 0.2% Triton X-100 (20 min, 4 °C). After three wash steps, unspecific binding was prevented by the incubation of cells in PBSo with 5% FCS/1% BSA for 1 h at 20 °C in a humid chamber. After this blocking step, cells were washed three times with PBSo/5% FCS/1% BSA. Subsequently, cells were stained with primary antibodies mouse anti-Tax (1:2) [87] and rabbit polyclonal anti-ELL2 (1:200; A302–505A, Bethyllabs) in PBSo/5% FCS/1% BSA (30 min, 37 °C in humid chamber) followed by washing and secondary antibodies anti-mouse AlexaFluor 647 and anti-rabbit AlexaFluor 488 in PBSo/5% FCS/1% BSA (1:200 each, Life Technologies, 1 h, humid chamber, 37 °C). After three final washing steps with PBSo, slides were covered with ProLong Gold antifade reagent with DAPI (Life Technologies) and analyzed by confocal laser scanning microscopy. All images were acquired using a Leica TCS SP5 confocal laser scanning microscope equipped with a 63 × 1.4 HCX PL APO CS oil immersion objective lens. LAS AF software (Leica, Wetzlar, Germany) was used for the analysis of images. To quantitate the frequency of Tax⁺ELL2⁺ cells in which ELL2 forms nuclear speckles with Tax, 120 Tax⁺ELL2⁺ cells in eight different optical fields were evaluated.