PMC:6218809 / 8260-10296
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6218809","sourcedb":"PMC","sourceid":"6218809","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6218809","text":"Interphase-, Metaphase-FISH, and Karyotyping by Multiplex-FISH\nMetaphase chromosomes were prepared from a human lymphoblastoid cell line (HG02554) purchased from Coriell Cell Repositories. Briefly, colcemid (Thermo Fisher Scientific) was added to a final concentration of 0.1 μg/mL for 1 hr, followed by treatment with hypotonic buffer (0.4% KCl in 10 mM HEPES [pH 7.4]) for 10 min and then fixed using 3:1 (v/v) methanol:acetic acid.\nFor interphase- and metaphase-FISH, G248P8211G10 labeled with Texas Red-dUTP, G248P85804F12 labeled with Atto488-XX-dUTP (Jena Bioscience), and RP11-325A24 labeled with Atto425-dUTP (Jena Bioscience) were used as probes. Slides pre-treatments included a 10-min fixation in acetone (Sigma-Aldrich), followed by baking at 65°C for 1 hr. Metaphase spreads on slides were denatured by immersion in an alkaline denaturation solution (0.5 M NaOH, 1.0 M NaCl) for 10 min, followed by rinsing in 1 M Tris-HCl (pH 7.4) solution for 3 min, 1× PBS for 3 min, and dehydration through a 70%, 90%, and 100% ethanol series. The probe mix was denatured at 65°C for 10 min before being applied onto the denatured slides. Hybridization was performed at 37°C overnight. The post-hybridization washes included a 5-min stringent-wash in 1× SSC at 73°C, followed by a 5-min rinse in 2× SSC containing 0.05% Tween20 (VWR) and a 2-min rinse in 1× PBS, both at room temperature. Finally, slides were mounted with SlowFade Gold mounting solution containing 4′6-diamidino-2-phenylindole (Thermo Fisher Scientific). Multiplex-FISH (M-FISH) with human 24-color painting probe, as previously described.34\nSlides were examined using AxioImager D1 microscope equipped with appropriate narrow-band pass filters for DAPI, Aqua, FITC, Cy3, and Cy5 fluorescence. Digital image capture and processing was carried out using the SmartCapture software (Digital Scientific UK). Ten randomly selected metaphase cells were karyotyped based on the M-FISH and DAPI-banding patterns using the SmartType Karyotyper software (Digital Scientific UK).","divisions":[{"label":"title","span":{"begin":0,"end":62}},{"label":"p","span":{"begin":63,"end":434}},{"label":"p","span":{"begin":435,"end":1609}}],"tracks":[{"project":"2_test","denotations":[{"id":"30388403-26388286-2049231","span":{"begin":1607,"end":1609},"obj":"26388286"}],"attributes":[{"subj":"30388403-26388286-2049231","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#9395ec","default":true}]}]}}