Anti sc35

Formalin‐fixed paraffin‐embedded 4‐μm thick tumour sections were subjected to CCND1 immunostaining, according to the manufacturer's instructions. The antibodies (CCND1a, 1:200; CCND1b, 1:150) were incubated overnight. Negative controls used non‐specific rabbit Immunoglobulin G instead of the primary antibody, and a BC pathologist performed a blind evaluation of the immunostained sections. Slides stained with CCND1a and CCND1b antibodies were evaluated and blinded by an independent BC pathologist. Tumours with >50% CCND1a staining were considered CCND1a positive. Since CCND1b is not generally found in normal cells, tumours were considered positive for CCND1b if any cells showed staining in the nucleus.²⁰, ³⁸, ³⁹ Cells were seeded on glass coverslips, fixed using either 3% paraformaldehyde and blocked for 1 h in 4% BSA/PBS followed by incubation in Anti‐SC35 (1:100, Abcam) for 1 h at room temperature. Cells were washed in PBS and incubated in a secondary FITC conjugated antibody (1:200) for 30 min at room temperature. Cells were washed with PBS and counterstained with Anti‐Lamin A (1:500, Abcam). The specimens were examined with a confocal laser microscope (LSM, Carl Zeiss AG).

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).