Axioplan 2 imaging fluorescence microscope

Cells were grown on coverslips in culture medium till 50–60% confluency before transfection or when 80–90% confluency reached for untreated cells. They were then fixed with 4% paraformaldehyde in PBS (freshly prepared and pre-warmed to 37°C) for 30 min at 37°C, washed thrice with 1XPBS before permeabilizing with 0.2% Triton X-100 in PBS on ice for 5 min. Alternatively, cells were fixed and permeabilized with methanol at −20°C for 7 min. After permeabilization, cells were washed thrice with 1XPBS and blocked with 5% bovine serum albumin (BSA) in PBS for 30 min. Cells were then incubated in primary antibodies at their appropriate dilutions for 2 h at room temperature and subsequently washed with PBS thrice, 5 min each followed by incubation with the appropriate Alexa Fluor secondary antibodies (Invitrogen, Carlsbad, CA, USA) with 1:200 dilution, for 1 h at room temperature in the dark. The cells were washed 3 times, 5 min each after the incubation. The nuclei of the cells were counterstained with 150 nM of 4,6-diamidino-2-phenylindole (DAPI) for 5 min. After washing the cells thrice with PBS, the coverslips with cells were mounted with SlowFade® Gold antifade reagent (Molecular Probes, CA, USA) before sealing with transparent nail polish. Images were subsequently visualized and captured using Axioplan 2 imaging fluorescence microscope (Carl Zeiss) (43 ).

FISH was performed to detect EWSR gene rearrangement using Vysis LSI EWSR1 Break Apart FISH probe kit (Abbott Molecular, Abbott Park, IL, USA) in accordance with the manufacturer’s recommended procedures as described previously [30 (link)]. With pepsin for 25 min at 37 °C, slides were deparaffinized and immersed in ethanol baths. After air_drying, hybridization was performed. After counterstaining with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI). Images were acquired with an Axioplan2 imaging fluorescence microscope (ZEISS, Göttingen, Germany) equipped with a CCD camera and appropriate filters. Images were subsequently analyzed with the digital-imaging software Isis v3.8.8 (MetaSystems, Altlussheim, Germany).
The orange and green probes used for this analysis labelled the 5’ and the 3’ site of the EWSR1 gene, respectively. Therefore, each allele of the EWSR1 gene will exhibit two contiguous orange and green signals in cells with intact 22q12 regions. On the contrary, in the presence of translocations related to the 22q12 region, the two signals will be split. We considered an allele positive for EWSR1 rearrangement when the gap between the green and orange signals exceeded the diameter of either one.

The cellular (co)-localization of DZANK1, NINL^isoB, DYNLL1 and DYNLL2 was determined by co-transfecting hTERT-RPE1 cells on glass slides, with pcDNA3-mRFP and pcDNA3-eCFP. DZANK1 FL, NINL^isoB, DYNLL1 and DYNLL2 were transfected using Effectene Transfection Reagent (Qiagen, Netherlands) according to manufacturer’s instructions. After 48 hours transfection, cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) and mounted with Vectashield containing DAPI (Vector Laboratories, Inc., UK). Images were taken with an Axioplan2 Imaging fluorescence microscope (Zeiss) and processed using Adobe Photoshop version 8.0 (Adobe Systems, USA).