Anti coilin

The method has been widely implemented by our laboratories. Cells of 5.0 × 10⁴ were plated onto a glass coverslip placed into the well of a 12-well plate. The cells on coverslips were fixed, permeabilized, blocked and washed with phosphate-buffered saline (PBS). Anti-coilin (Proteintech Group, Rosemont, USA) was used as the primary antibody and an Alexa Fluor® 594 secondary antibody (Life Technologies, MA, USA) was used for incubation in the dark. The nuclei were stained with Hoechst 33,258 (Sigma-Aldrich, St. Louis, MO, USA) prior to the examination and image acquisition under a confocal system (Leica Microsystems TCS SP8. Wetzlar, Germany). Control samples without adding the primary antibody were prepared for determining the level of non-specific noise.

HeLa cells, treated either with DMSO or compound, were grown on coverslips in DMEM for 24 h at 37 °C before fixing with 4% paraformaldehyde in PHEM buffer for 10 min at room temperature. After rinsing the cells with PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS prior to incubation with the primary antibodies, i.e. anti-SC35 (Abcam, Cambridge, UK), anti-coilin (ProteinTech, Chicago, IL), anti-Y12 (Dundee Cell Products, Dundee, UK), anti-TMG (Calbiochem), and anti-SMN (BD Biosciences). After incubation with the primary antibody for 1 h at room temperature, the coverslips were washed twice with 0.5% of Tween 20 in PBS for 5 min before they were incubated with the dye-conjugated secondary antibody for 30 min. Cells were then stained with DAPI (Sigma), and the coverslips were mounted in Vectashield medium (Vector Laboratories, Peterborough, UK). The samples were visualized using a fluorescence microscope (Zeiss, Jena, Germany; Axiovert-DeltaVision Image Restoration; and Applied Precision, LLC).