Anti digoxigenin fluorescein fab fragments

The five different karyotypes (Types I–V) were used for whole-chromosome painting (WCP). The painting probes were labeled with Dig-11-dUTP (Roche Diagnostics, Germany) via whole-genome amplification (WGA: WGA3 kit, Sigma, USA). Painting fluorescence in situ hybridization (FISH) was accomplished following the protocol of Yang et al. (1995) (link) with minor modifications. To block highly repetitive DNAs, C₀t-1 DNA from Q. boulengeri was made according to the procedure described by Zwick et al. (1997) (link). The hybridization mix contained 5 ng/μL probe DNA, 40 ng/μL C₀t-1 DNA, 10% dextran sulphate, and 50% deionized formamide in 2× SSC, denatured at 100°C for 10 min and pre-annealed by incubation at 37°C for 80 min. After hybridization overnight in a wet chamber at 37°C, the probes were detected with anti-digoxigenin-fluorescein fab fragments (Roche, Germany). Chromosomes were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, Vector). Hybridization signals were observed under a Leica DM2500 fluorescence microscope equipped with a fluorescent lamp and an appropriate filter set.

Prior to hybridization, chromosomes were Q-banded using 0.005% quinacrine mustard solution for 90 s. A standard FISH protocol was applied65 (link), using three genomic probes: hCD59 labelled with DEAC-5-dUTP (PerkinElmer, USA), hCD55 with dig-11-dUTP (Roche, Germany), and hCD46 with biotin-16-dUTP (Roche, Germany). In each case mean probe fragment size was ~500 bp. Digoxigenin-labelled probes were detected with anti-digoxigenin-fluorescein Fab fragments (Roche). Chromosome preparations were mounted in Vectashield (Vector Laboratories) with DAPI counterstain and analysed under a Nikon Ni-U fluorescence microscope. International nomenclature for identification of pig chromosomes was applied66 (link).

Plasmid pSFVdhfr was used to prepare a probe for detection of the plasmid sequence in clone 12 (DM) and clone 22 (HSR) cells [5 (link)]. Cosmid DNA (c-myc) was used to detect natural amplicons in COLO 320DM and COLO 320HSR cells [6 (link)]. Probes were DIG- or biotin-labeled using the BioPrime DNA Labeling System (Invitrogen) with or without 10×DIG DNA Labeling Mixture (Roche Lifescience Inc.). The probe was hybridized to metaphase chromosome spreads, and hybridized DIG- and biotin-labeled probes were detected using anti-Digoxigenin-Fluorescein Fab fragments (Roche) or Streptavidin Alexa Fluor 594 conjugate (Invitrogen), respectively.

Fibroblast cell cultures were established in DMEM medium (Gibco, Life Technologies, Carlsbad, CA, USA) with nonessential aminoacids (Sigma-Aldrich, St. Louis, MO, USA), penicillin/streptomycin and 10% fetal calf serum, from tail samples from transgenic mice. The cells were grown for 10–14 days and then harvested following colcemid inhibition of cell division for 3–6 h. Chromosome preparations were obtained by using standard techniques. A probe was prepared from the intact T9W-IVSI-6 vector, directly labeled by nick translation with the DIG-Nick Translation Mix (Roche Applied Science, Penzberg, Upper Bavaria, Germany) according to the manufacturer's protocol. The probe was hybridized and then detected with anti-digoxigenin-fluorescein Fab fragments (Roche Applied Science). The slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) containing 4′,6-diamidino-2-phenylindole (DAPI) counterstain. FISH signals were examined with Olympus Provis epifluorescence microscope and images were captured using Leica Microsystems CytoVision imaging equipment and software (Applied Imaging, Leica-Microsystems, Wetzlar, Germany). The chromosomal site of transgene integration was determined by karyotypic analysis of banded chromosomes obtained using the DAPI image.

Metaphase chromosome spreads were prepared by a standard protocol [21 ]. Chromatin fibers were prepared according to our previously published protocol [5 (link)], which was developed based on the preceding protocol [22 (link)]. DIG-labeled plasmid probes were prepared from pG5 or pKV-AR1 plasmid DNA using the BioPrime DNA Labeling Kit (Invitrogen) and 10× DIG DNA Labeling Mixture (Roche). Biotin-labeled G5 probe was prepared by PCR amplification of the G5 sequence (966 bp) in the pG5 plasmid using the primer set used for the preparation of repeat DNA (see above) and biotin labeling of the product using the Biotin PCR labeling kit (Promokine). The probe was hybridized to metaphase chromosome spreads, and hybridized DIG- and biotin-labeled probes were detected using anti-Digoxigenin-Fluorescein Fab fragments (Roche) or Streptavidin Alexa Fluor 594 conjugate (Invitrogen), respectively. For the flow cytometric analysis to evaluate d2EGFP expression, the cells were resuspended in phosphate buffered saline and analyzed using FACS Calibur (Becton Dickinson Co.).

Hsa-miR-29c expression in liver carcinoma specimens (LVC481 and LVC2281 tissue arrays; Pantomics, Richmond, CA) was determined by FISH, as detailed elsewhere [63 (link)]. After deparaffinization, the sections were prehybridized for 20 minutes at 55°C followed by 1 hour hybridization at the same temperature with a 1:1000 dilution of a miRCURY LNA™ hsa-miR-29c detection probe (Exiqon, Vedbaek, Denmark). After washing, the sections were blocked for 1 hour with a blocking solution and incubated with a 1:1000 dilution of anti-Digoxigenin-Fluorescein, Fab fragments (Roche, Basel, Switzerland) at 4°C overnight. Then research scientists and a pathologist independently analyzed the stained tissue sections.
Staining intensity was the criterion used for quantitating immunofluorescence staining. A range of 0 to 3 was used for classifying the intensity: 0 = absence of staining; 1 = weak staining; 2 = moderate staining; and 3 = intense staining.