CBG-banding was obtained according to Sumner.29 The Callithrix, Callimico and M. argentatus karyotypes were mounted following Sherlock et al.,30 (link) Neusser et al.31 (link) and Dumas et al.,32 respectively. Fluorescence in situ hybridization (FISH) was performed using AS, MarmoSAT and telomeric sequences as probes. The satDNA probes were prepared from PCR purified products labelled by nick translation with digoxigenin-11-dUTP (DIG-Nick Translation mix, Roche Applied Science). A biotinylated telomeric sequence (TTAGGG)4 (Invitrogen) was synthesized and used as probe for FISH. Chromosomes were denatured in 70% formamide/2xSSC at 65% for 1–2 min. The hybridization mix, consisting of 100 ng of labelled probe in 50% formamide/2xSSC, was denatured for 10 min at 98°C and applied to the chromosome preparations. Hybridization was carried out at 37°C for 16–20 hours. Slides were washed in 2xSSC at 37°C for 5 min. Immunodetection was performed with antidigoxigenin conjugated with FITC and neutravidin coupled with rhodamine (Roche Applied Science). The analyses and image acquisition were performed under a Zeiss Axioimager 2 epifluorescence microscope using the AxioVision software (Zeiss).
Rhodamine
Manufactured by Roche
Sourced in Germany
Rhodamine is a fluorescent dye commonly used in laboratory applications. It exhibits bright red-orange fluorescence when excited by light. Rhodamine is a useful tool for various analytical and imaging techniques due to its strong fluorescence properties.
Automatically generated - may contain errors
Lab products found in correlation
Cot 1 dna, by Roche (2 mentions)
Isis digital image analysis software system, by MetaSystems (2 mentions)
Fluorescein isothiocyanate (fitc), by Vector Laboratories (2 mentions)
Axio imager 2, by Zeiss (1 mentions)
Dig nick translation mix, by Roche (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Dig dutp, by Roche (1 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Anti dig antibody, by Roche (1 mentions)
Digoxigenin 11 dutp dig dutp, by Roche (1 mentions)
Fluorescein isothiocyanate (fitc), by Roche (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Antifade solution, by Vector Laboratories (1 mentions)
Human cot 1 dna, by Thermo Fisher Scientific (1 mentions)
Digoxigenin 11 dutp, by Roche (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Chroma Technology (1 mentions)
Anti digoxigenin antibody, by Roche (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Bx61 microscope, by Olympus (1 mentions)
7 protocols using rhodamine
1
Karyotyping and FISH Analysis of Callitrichidae
2
Comparative Genomic Hybridization Protocol
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CGH was performed as previously described [27 (link)]. Briefly, DNA from UM-SCC1 cells and reference DNA from the blood of a healthy donor were obtained using standard phenol/chloroform extraction, labeled with biotin and digoxigenin by nick translation according to the manufacturer's protocol (Roche Diagnostics, Mannheim, Germany). Hybridization was performed together with COT-1 DNA (Roche Diagnostics) to normal chromosome metaphase spreads from peripheral blood lymphocytes prepared using the following standard procedures. Post-hybridization washes were performed with 50% formamide/2× standard saline citrate (SSC), 2× SSC and 0.1 × SSC at 45°C. DNA was visualized with fluorescein-isothiocyanate (FITC, Vector Laboratories, Burlingame, CA) and rhodamine (Roche Diagnostics), respectively, and counterstained with a DAPI (4, 6-diamidino-2-phenylindole) anti-fade solution (Vector Laboratories). Fluorescence images were captured using a fluorescence microscope Olympus BX 61 and image processing was performed using the ISIS digital image analysis software system (MetaSystems, Altlussheim, Germany). Average ratio profiles were determined from the analysis of 12–15 metaphases.
3
Double DNA Labeling and Detection
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Cells were labeled with 100 µM EdU (Life Technologies, Carlsbad, CA, USA) for 10 min, aliquoted onto coverslips and fixed with 4% paraformaldehyde in PBS, and then permeabilized with 0.5% Triton X-100 in PBS. Blocking was performed by incubation in 3% BSA/0.1% Tween 20/Tris-buffered saline, pH for 30 min. EdU-labeled DNA was detected with the Click-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Digoxigenin (DIG)-labeled DNA was detected with an anti-DIG antibody conjugated with FITC (1:200 dilution; Roche, Mannheim, Germany). DAPI was used to counterstain the DNA. In the experiment in Figure 1 B, digoxigenin-11-dUTP (DIG-dUTP; Roche, Mannheim, Germany) was loaded into cells with the hypotonic shift method for replication labeling [37 (link)]. Incorporated DIG-dUTP was detected with an anti-DIG antibody conjugated with rhodamine (Cat#11207750910; Roche, Mannheim, Germany).
4
Metaphase Chromosome Spread Analysis
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Metaphase chromosome spreads were generated from the H2AX8 and derivative cell lines using conventional cytogenetic techniques and subsequently pretreated and denatured as described elsewhere (Roschke et al, 2002). BAC DNA was labeled by nick translation with digoxigenin-11-dUTP or biotin-16-(Roche Applied Science), precipitated in ethanol with 50 × excess of human Cot-1 DNA (Invitrogen), and resuspended to a final concentration of 50 ng/μl in Hybrizol solution (MP Biomedicals). Whole chromosome painting probes for were obtained from Applied Spectral Imaging. After denaturing at 80 °C for 10 min and preannealing at 37 °C for 60 min, 10 μl of probe mixtures were applied under 22 mm × 22 mm coverslips. Slides were incubated in a moist chamber overnight at 37 °C. After detection with anti-digoxigenin antibodies labeled with rhodamine (Roche Applied Science) or avidin conjugated with FITC (Roche Applied Science) they were mounted in antifade solution (Vector Laboratories) containing DAPI. A Leica microscope equipped with DAPI, FITC and rhodamine filters (Chroma Technology) and a Sensys CCD camera (Photometrics) connected with Q-FISH software (Leica Microsystems Imaging Solutions) were used for image acquisitions.
5
PcP190 Satellite DNA Probe Labeling
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The labeling of the isolated PcP190 satellite DNA probes used in this analysis was based on PCR amplification in the presence of Digoxigenin-11-dUTP with a DIG Probe Synthesis PCR (Roche, Pensberg, Bavaria, Germany). The probes were mixed with salmon DNA (1 ng/μL of probe) and precipitated with ethanol. All the resulting DNA was dissolved in a hybridization buffer at pH 7 composed of deionized formamide (50%), 2x SSC, phosphate buffer (40 mM), Denhardt’s solution, SDS (1%) and dextran sulfate (10%).
The hybridization method used was that described by Viegas-Péquignot (1992) , with adaptations for the detection of the Digoxigenin-11-dUTP, which was based on the anti-digoxigenin antibody conjugated with rhodamine (Roche, Pensberg, Bavaria, Germany).
The hybridization method used was that described by Viegas-Péquignot (1992) , with adaptations for the detection of the Digoxigenin-11-dUTP, which was based on the anti-digoxigenin antibody conjugated with rhodamine (Roche, Pensberg, Bavaria, Germany).
6
Fluorescence In Situ Hybridization Protocol
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Chromosome spreads were subjected to FISH using the constructed probes. FISH was
performed under a high stringency of approximately 76% (2.5 ng/μL of each probe,
50% formamide, 2 SSC, 10% dextran sulfate, pH 7.0–7.2, 37 °C overnight)
following the general procedure described by Pinkel et al. (1986) (link). Signal detection was
performed using an anti-streptavidin antibody conjugated to Alexa Fluor 488
(Molecular Probes, Eugene, OR, USA) and an anti-digoxigenin antibody conjugated
to rhodamine (Roche Applied Science). Chromosomes were counterstained with
4'6-diamidino-2-phenylindole (0.2 μg/mL) in Vectashield mounting medium (Vector
Laboratories, Burlingame, CA, USA) and observed under an epifluorescence
microscope.
performed under a high stringency of approximately 76% (2.5 ng/μL of each probe,
50% formamide, 2 SSC, 10% dextran sulfate, pH 7.0–7.2, 37 °C overnight)
following the general procedure described by Pinkel et al. (1986) (link). Signal detection was
performed using an anti-streptavidin antibody conjugated to Alexa Fluor 488
(Molecular Probes, Eugene, OR, USA) and an anti-digoxigenin antibody conjugated
to rhodamine (Roche Applied Science). Chromosomes were counterstained with
4'6-diamidino-2-phenylindole (0.2 μg/mL) in Vectashield mounting medium (Vector
Laboratories, Burlingame, CA, USA) and observed under an epifluorescence
microscope.
7
Comparative Genomic Hybridization Protocol
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CGH was performed as described previously (24) . Briefly, genomic DNA from FaDu cell line and reference DNA from blood of a healthy donor were obtained using standard phenol/chloroform extraction, labeled with biotin and digoxigenin by nick translation according to the manufacturer's protocol (Roche Diagnostics). Hybridization was performed together with COT-1 DNA (Roche Diagnostics) to normal chromosome metaphase spreads from peripheral blood lymphocytes prepared using standard procedures. Post-hybridization washes were performed with 50% formamide/2× standard saline citrate (SSC), 2× SSC and 0.1× SSC at 45°C. DNA was visualized with fluorescein-isothiocyanate (Vector Laboratories) and rhodamine (Roche Diagnostics), respectively, and counterstained with a DAPI (4, 6-diamidino-2phenylindole) anti-fade solution (Vector Laboratories). Fluorescence images were captured using an Olympus BX61 microscope (Olympus) and image processing was performed using the ISIS digital image analysis software system (Metasystems). Average ratio profiles were determined from the analysis of 12-15 metaphases. The heterochromatic regions in chromosomes 1, 9 and 16, the p-arms of the acrocentric chromosomes, and Y chromosome were excluded from the analysis because of suppression of hybridization with COT-1 DNA in these regions.
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