Nick translation kit

TRAC, B2M, and PDCD1 DNA probes were labeled by nick translation reaction (Nick Translation Kit—Abbott Molecular) using Green—500 dUTP (Enzo Life Science). Sizes of the nick translated fragments were checked by electrophoresis on a 1% TBE gel. The labeled DNA was precipitated in COT-1 DNA, salmon sperm DNA, sodium acetate and 95% ethanol, then dried and resuspended in 50% formamide hybridization buffer. The probe/hybridization buffer mix and slides were denatured, probe was applied to the slides, and slides were hybridized for 48 h at 37° in a humidified chamber. After hybridization, the FISH slides were washed in a 2× SSC solution at 72° for 15 s, and counterstained with DAPI. Fluorescent signals were visualized on an Olympus BX61 microscope workstation (Applied Spectral Imaging, Vista, CA) with DAPI and FITC filter sets. FISH images were captured using an interferometer-based CCD cooled camera (ASI) and FISHView ASI software.

Cells were plated on either slides or coverslips. After washing twice with PBS, cells were fixed for 10 min with 2.6% formaldehyde followed by permeabilisation with 0.4% Triton X-100 for 5 min at 4°C. After a quick PBS wash, cells were incubated with probes in a humid chamber overnight at 37°C. Xist RNA probes were generated from an 18 kb fragment spanning the whole Xist transcript using a nick translation kit (Abbott Molecular) as previously described (Moindrot et al., 2015 (link)). Labeled RNA probes (1.5 μL) were co-precipitated with 10 μg salmon sperm DNA, 1/10 volume 3 M sodium acetate (pH 5.2) and 3 vol ethanol. After washing in 75% ethanol, the pellet was dried, resuspended in 6 μL formamide and denatured at 75°C for 7 min before flash cooling on ice. Probes were diluted in 6 μL 2x hybridization buffer (5x SSC, 12.5% dextran sulfate, 2.5 mg/mL BSA (NEB)), added to the slide/coverslips and incubated overnight at 37°C in a humid chamber. After incubation, slides/coverslips were washed three times with a solution of 2xSSC/50% formamide followed by three washes with 2xSSC in a water bath at 42°C. Slides/coverslips were mounted and sealed as for immunofluorescence.

The following were from commercial sources: Trizol™ reagent, Superscript™ First-Strand Synthesis System for RT-PCR Kit, NuPAGE 4–12% Bis-Tris gel, and NuPAGE MES SDS running buffer, N-6-[(7-nitrobenzo-2-oxa-1, 3-diazol-4-yl) amino] hexanoyl-4-d-erythro-sphingosine (C6-NBD-ceramide), Alexa-Fluor555 (Invitrogen, Carlsbad, CA); anti-β-actin monoclonal antibody (Sigma, St. Louis, MO); rat anti-mouse CD68 monoclonal antibody (Serotec, Oxford, UK); M-PER Mammalian Membrane Protein Extraction Reagent and BCA Protein Assay Reagent (Pierce, Rockford, IL); [32P] dCTP (DuPont-New England Nuclear Research Products, Boston, MA); Molecular Dynamics Storm 860 scanner, Hybond™-ECL™ nitrocellulose membrane, and ECL detection reagent (Amersham Biosciences, Piscataway, NJ); antifade/4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratory, Burlingame, CA); FITC-conjugated goat anti-rabbit antibody and rhodamine-conjugated goat anti-rat antibodies (ICN Biomedicals, Aurora, OH); pBROAD3-mcs vector carrying ubiquitous murine ROSA26 promoter (InvivoGen, San Diego, CA); Genomic BAC probe RP23 (BACPAC Resources Center, Oakland CA); Nick Translation Kit (Abbott Molecular, Des Plaines, IL); Hybond N+ nylon membranes (Amersham Bioscience, Freiburg, Germany).

Insect telomeric probe (TTAGG)_n was synthesized by means of non-template PCR as described previously [47 (link)] and labeled with Cy3-dUTP or fluorescein-12-dUTP either by Nick Translation Kit (Abbott Molecular; for details, see above) or using the improved nick translation procedure [48 (link)] with some modifications. The modified 20 µL reaction contained 1 µg unlabeled DNA; 50 µM dATP, dCTP, and dGTP; 10 µM dTTP; 20 µM labeled nucleotides; 1× nick translation buffer (50 mM Tris-HCl, pH 7.5; 5 mM MgCl₂; 0.005% BSA); 10 mM β-mercaptoethanol; 0.005 U DNase I and 20 U DNA polymerase I (both Thermo Fisher, Waltham, MA, USA). The reaction was incubated at 15 °C for 1 h.
An 18S ribosomal DNA (rDNA) probe was generated by PCR from the codling moth (Cydia pomonella) gDNA [49 (link)] and labeled with biotin-16-dUTP (Roche Diagnostics, Mannheim, Germany) by improved nick translation procedure (for details, see above). The reaction was incubated at 15 °C for 1 h.

FISH was performed using the Histology FISH Accessory kit (DAKO, Glostrup, Denmark) on 5-μm sections from FFPE samples, as recommended by the manufacturer. The probes consisted of a chromosome-17 centromeric probe (CEP17 Spectrum Green ™ probe, Vysis, Abbott Molecular, Des Plaines, Illinois, USA) and an in-house generated bacterial artificial chromosome (BAC) probe located in the amplified region of chromosome 17 (clone RP11-1055B8). The BAC probe was labeled with SpectrumOrange™ using a nick translation kit (Abbott Molecular, Des Plaines, Illinois, USA) and the specificity of the BAC probe was verified on leucocytes by metaphase FISH.
Images were collected with the BioView Duet™ System (BioView Ltd, Rehovot, Israel). Signals from both probes were scored in about 100 non-overlapping nuclei with malignant morphology in the infiltrating tumor zone (as selected by the pathologist). Adjacent fibroblasts or lymphocytes and residual normal breast tissues were used as internal control. For each tumor, the mean number of signals corresponding to the RP11-1055B8 probe was calculated. The ratio between the RP11-1055B8 probe and the centromeric probe was also evaluated for individual nuclei and the percentage of the nuclei with a ratio ≥3 was calculated for each TNBC. FISH slides were analyzed blinded to the CGH results.