Roots from Q. faginea, Q. pyrenaica, Q. suber, F. sylvatica, and C. sativa were treated to induce C-metaphases and chromosome spreads according to Ribeiro et al [22] . The DNA fluorescent in situ hybridization (FISH) technique was adapted from [40] , with a stringency of 74% and post-hybridization washes with a stringency of 84%. The Beech NTS-5′-ETS labeled by nick translation with biotin-dUTP (Roche, Switzerland) was simultaneously hybridized with pTa71, a 9-kb fragment of rDNA from wheat [41] (link) also labeled by nick translation with digoxigenin, on metaphase chromosomes of F. sylvatica, Q. suber, C. sativa, and Q. pyrenaica, while Cork Oak NTS-5′-ETS, labeled by nick translation with biotin-dUTP (Roche, Switzerland) and pTa71 were used in species of the three contrasting genera: F. sylvatica, Q. suber, and C. sativa. DNA was counterstained with VectaShield Mounting Medium with DAPI (Vector Laboratories, USA). Measurements of the IGS and pTa71 fluorescent signal intensity were performed in ten cells (from two different individuals), using the AxioVision measurement module of epifluorescence microscope Axio Imager.Z1 (Zeiss, Germany).
Biotin dutp
Manufactured by Roche
Sourced in Switzerland, Germany, United States
Biotin-dUTP is a nucleotide analog that contains a biotin molecule attached to the uracil base. It can be incorporated into DNA during synthesis, allowing for the detection and labeling of DNA sequences. The biotin moiety serves as a tag that can be identified and visualized using various detection methods, such as streptavidin-based techniques.
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Lab products found in correlation
Digoxigenin dutp, by Roche (10 mentions)
Endogenous biotin blocking kit, by Roche (2 mentions)
Ddh2o, by Thermo Fisher Scientific (2 mentions)
Ldh cytotoxicity detection kit, by Takara Bio (1 mentions)
Vectashield antifade solution, by Vector Laboratories (1 mentions)
Axiovision measurement module, by Zeiss (1 mentions)
Szx16 microscope, by Olympus (1 mentions)
Tdt buffer, by Merck Group (1 mentions)
Streptavidin conjugated horseradish peroxidase, by Roche (1 mentions)
Saponin, by Merck Group (1 mentions)
Rhodamine conjugated anti digoxigenin, by Roche (1 mentions)
Fluorescein conjugated avidin, by Thermo Fisher Scientific (1 mentions)
Cellsens dimension software, by Olympus (1 mentions)
Bx63 fluorescence microscope, by Olympus (1 mentions)
Streptavidin hrp and dab detection kit, by Roche (1 mentions)
Avidin alexa 488, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
Ex taq buffer, by Takara Bio (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
34 protocols using biotin dutp
1
Comparative FISH Analysis of Fagaceae
2
Fluorescent in-situ Hybridization of Brassica Genomes
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Preparation of slides and hybridization were carried out according to procedures detailed by Suay et al. (2014) (link). The ribosomal probe used in this study was 35S rDNA (pTa 71 clone) from wheat (Gerlach and Bedbrook, 1979 (link)), IGS-A and IGS-C probes described further below and the BAC clone B. oleracea named Bob014O06 (Howell et al., 2002 (link)). This BAC clone was used as ‘GISH-like’ to distinguish specifically all C-genome chromosomes in B. napus (Suay et al., 2014 (link)). The 35S rDNA and BAC clone were labelled with Alexa-488 dUTP by random priming, the IGS-A with biotin-dUTP (Roche, Mannheim, Germany) using PCR and the IGS-C with biotin-dUTP (Roche) using nick translation (Bionick DNA labelling System, Thermo Fisher Scientific, Waltham, MA, USA). Biotinylated probe was immunodetected by Texas Red avidin DCS (Vector Laboratories, Burlingame, CA, USA) and the signal was amplified with biotinylated anti-avidin D (Vector Laboratories). The chromosomes were mounted and counterstained in Vectashield (Vector Laboratories, Ontario, Canada) containing 2·5 μg mL–1 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, AZ, USA) on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analysed using MetaVueTM (Universal Imaging Corporation, Downington, PA, USA).
3
Amplification and Labeling of Repeat Families
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The rye 120-bp repeat family (Bedbrook et al., 1980 (link)) was amplified by PCR from DNA clone pSc119.2 using M13 universal primers (Said et al., 2018 (link)), whereas Afa family repeat was amplified from genomic DNA of bread wheat CS using primers AS-A and AS-B (Nagaki et al., 1995 (link)). The probes pSc119.2 and Afa family repeat were labeled by PCR with digoxigenin-dUTP and biotin-dUTP (Roche, Mannheim, Germany), respectively (Said et al., 2018 (link); Said et al., 2019b (link)). Plasmid pTa71 (45S rDNA) containing a 9-kb fragment from T. aestivum with 18S-5.8S-26S rDNA and intergenic spacers (Gerlach and Bedbrook, 1979 (link)) was labeled by nick translation with either biotin-dUTP or digoxigenin-dUTP (Roche) using standard Nick Translation Mix kits (Roche) following the instructions of the manufacturer.
4
Quantifying Islet β-Cell Death In Vitro and In Vivo
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For detection of islet/β-cell death in vitro, cells were exposed to hypoxia (1% O2) for 24 or 48 h. Cells were harvested and washed with PBS containing 5 mM EDTA. After fixation in 70% ethanol at 4 °C for 2 h, cells were collected, re-suspended in PBS containing 250 μg/ml RNase A, and incubated at 37 °C for 30 min. After propidium iodide staining, 2 × 105 cells per sample were analyzed by flow cytometry. The portion of the apoptotic cells was calculated according to the apoptotic peak in the cell distribution against the propidium iodide fluorescence per cell. Cell death was also measured using a Cell Death Detection ELISA Kit and the colorimetric assay in medium using a LDH cytotoxicity detection kit (Clontech) as suggested by manufacturer (Roche, Indianapolis, IN, USA). For detection of β-cell apoptosis inside the pancreas, pancreatic tissue sections were incubated with terminal deoxynucleotidyl transferase in the presence of Biotin-dUTP (Roche Diagnostics). TUNEL-positive cells were revealed with Alexa Fluor 488-Streptavidin. Sections were double stained for insulin to distinguish β cells. Images were obtained by confocal microscopy. The numbers and fraction of TUNEL+ cells within an islet were calculated.
5
Cytogenetic Analysis of Sugarcane Genotypes
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The chromosomal preparations and the FISH technique were conducted as described in D'Hont et al [6 ]. Root tips were obtained from Yunnan82-114 (S. spontaneum), 51NG3 (S. robustum), and Luohanzhe (S. officinarum). The IGS labeled by nick translation with biotin-dUTP (Roche, Switzerland) was simultaneously hybridized with pTa71, and a 9-kb fragment of rDNA from wheat was also labeled by nick translation with digoxigenin on metaphase chromosomes of the above three materials [41 (link)]. High stringency conditions of post-hybridization washes were carried out with 2 × SSC for 5 min at 42°C, a second wash in 50% formamide, 2 × SSC, pH 7.0, for 3 × 5 min at 42°C, followed by a rinse in 2 × SSC for 5 min at room temperature and a final wash in 0.1 × SSC for 3 × 5 min at 55°C. Low stringency conditions were performed in 1 × SSC for 30 min at 37°C. Chromosomes was counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) in a Vectashield anti-fade solution (Vector Laboratories, Burlingame, CA). Measurements of the IGS and pTa71 fluorescent signal intensity were performed in at least in ten cells (from two different individuals) using the AxioVision measurement module of the AxioScope A1 Imager fluorescent microscope (Zeiss, Germany).
6
Apoptosis Evaluation via TUNEL Assay
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A TUNEL assay was performed by washing 4% paraformaldehyde-fixed cells on a coverslip once with PBS, followed by permeabilization using 0.5% saponin (Sigma-Aldrich) at room temperature for 30 min. Following a wash with terminal deoxynucleotidyl transferase (TdT) buffer (Roche Diagnostics, Indianapolis, IN, USA), cells were incubated with 0.5 µM biotin dUTP (Roche Diagnostics) and 150 U/ml of TdT (Sigma-Aldrich) in 30 µl of TdT buffer in a humidified chamber at 37°C for 30 min. Following two PBS washes, the cells were incubated with a 1/1,000 solution of streptavidin-conjugated horseradish peroxidase (Roche Diagnostics) in PBS for 10 min at room temperature. Coverslips were then washed for 30 min with three subsequent washes of PBS. Color was developed using TrueBlue peroxidase substrate (KPL, Inc., Gaithersburg, MD, USA), and coverslips were observed under an Olympus SZX16 microscope.
7
Centromeric Repeat Sequence Analysis
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FISH and fiber-FISH were carried out according to published protocols41 (link)61 (link)62 (link). The DNAs labeled with digoxigenin-dUTP (Roche Diagnostics, USA) and Biotin-dUTP (Roche Diagnostics, USA) were detected using rhodamine-conjugated anti-digoxigenin (Roche Diagnostics, USA) and fluorescein-conjugated avidin (Life Technologies, USA), respectively. DNAs were labeled with digoxigenin-dUTP and Biotin-dUTP for FISH analysis. Slides were examined under Olympus BX63 fluorescence microscope (Olympus, Japan). Chromosome and signal images were captured and merged using CellSens Dimension software (Olympus, Japan). Fiber-FISH was conducted to reveal the organization of the centromeric repeat sequences in SES208 genome. The fiber-FISH signals were measured and converted into kb using a 3.21-kb/μm conversion rate41 (link).
8
TUNEL Analysis Protocol for Apoptosis Detection
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TUNEL analysis was performed as follows. Slides were manually de-paraffinized in xylene, rehydrated in a series of alcohol dilutions (100%, 95%, and 70%) and tap water, and placed into the autostainer. Tissue sections were treated with Proteinase K (20 μg/mL in PBS) for 8 min, incubated with endogenous biotin blocking kit (Roche Diagnostics, Florham Park, NJ, USA) for 12 min, and incubated with labeling mix: TdT (Roche, 1000 U/mL) and biotin-dUTP (Roche, 4.5 nmol/mL) for 2 h. Detection was performed with Streptavidin—HRP and DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instruction.
9
3D-FISH Hybridization and Detection Protocol
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For 3D-FISH, the hybridization and detection protocol described by Solovei22 (link) was followed. All DNA probes were labeled in the presence of Biotin-dUTP (Roche) or TexasRed- dUTP (Molecular Probes), respectively, and mixed with 10-fold excess of human Cot-1 DNA. In situ hybridization was performed for 48 h, followed by stringency washes for 3 × 5 min in 0.1 × SSC (60°C). Biotinylated probes were detected with Avidin-Alexa 488 (Molecular Probes). Metaphase chromosomes and 3D fixed interphase nuclei were counterstained for 10 min with DAPI (2 μg/mL).
10
Genomic DNA Extraction and Probe Labeling
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For GISH, total genomic DNA (gDNA) was extracted from young leaves of seedlings using the cetyltrimethylammonium bromide (CTAB) based method described by Murray and Thompson [42 (link)]. DNA quality was evaluated by electrophoresis in 1 % agarose gel. DNA concentration was estimated using the ultraviolet spectrophotometer. About 1 ug gDNA of each species was used for probe labeling. For repeat DNA probes, Type III repeat of with the size of 177 bp (GenBank accession no. 18287), and CentM repeat of melon with the size of 352 bp (GenBank accession no. 3929695) were used for identification of centromeres of C. sativus and C. melo, respectively. Plasmids pTa71 with the size of 9 kb from wheat [43 (link)] was used to detect the 45S ribosomal DNAs. Type I/II repeat with the size of 182 bp (GenBank accession no. 18285) and Type IV repeat with the size of 360 bp (GenBank accession no. 18288) were also used for comparative mapping. The Arabidopsis-type telomere DNA was generated by polymerase chain reaction method in the absence of template using primers (TTTAGGG)4 and (CCCTAAA)4 according to Ijdo et al. [44 (link)]. The gDNA and all repeat DNA probes were labeled with either biotin-dUTP or digoxigenin-dUTP (Roche, http://www.roche-applied-science.com ) by standard nick translation reaction.
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