Chromosome preparation and Fluorescence in-situ hybridizati

Thursday, October 16, 2003

Description
Chromosome preparation and Fluorescence in-situ hybridization (FISH)

Procedure
僘?To be read as micro (mu)

1. Thin slides were kept in chromic acid overnight.
2. The slides were rinsed in running tap water for 30 minutes followed by scrubbing for 5 minutes with a gauze piece under running tap water.
3. Subsequently the slides were boiled in water and soap for 15 minutes.
4. After rinsing in water, the slides were stored at 4 刟C for 1 month.
5. The fixed lymphocytes were centrifuged at 1250 x g for 10 minutes and resuspended in 1-2ml of fresh fixative.
6. 7-8 drops of the suspension was dropped onto a chilled, precleaned slide drop by drop. The slide was gently heated and mouth-blown lightly to obtain a better spread.
7. After the slides had air-dried, they were kept at 4 刟C for 1 week for ageing.

FISH

1. Normal human chromosome spreads were prepared according to standard cytogenetic methodology .
2. Slides were treated with RNase (100僘磄/ml) [Sigma R-9009] for 45 minutes at 37 刟C in a moist chamber.
3. Slides were washed once in PBS and treated with pepsin (0.008%) [Sigma P-7012] for 10 minutes at 37 刟C.
4. Slides were passed through an ethanol series (70%, 85% and 100%) for 3 minutes each and stored at ?20 刟C till use.
5. 20ng of labeled probe DNA was denatured in a solution containing 13僘磍 formamide (68%) [Sigma F-7503], 1僘磍 of 2X SSC (0.1X), 3僘磍 of 10% dextran sulphate (1.6%) and 1僘磍 of 10僘磄/僘磍 calf thymus DNA (0.52僘磄/僘磍) [Sigma D-4522] for 5 minutes at 70 刟C. Slides containing target chromosomal DNA were denatured in 70% formamide [Sigma F-7503] and 2X SSC for 8 minutes at 70 刟C followed by quenching in a cold ethanol series as above.
6. The denatured probe was applied to the slide and allowed to hybridize overnight to two days at 37 刟C in a moist chamber.
7. Stringency washing was done in 50% formamide [Sigma F-7503] and 2X SSC for 10 minutes at 37 刟C; 2X SSC thrice for 4 minutes each at 37 刟C; and in 2X SSC with 0.1% Nonidet P40 (ethylphenylpolyethyleneglycol) [Fluka 74385] for 5 minutes at 37 刟C.
8. Slides were blocked in 2.5% blocking reagent in 2X SSC for 5 minutes at room temperature.
9. The slide was incubated with mouse anti-DIG monoclonal primary antibody [Boehringer Mannheim 1333062] (40僘磄/ml) for 45 minutes at 37 刟C in a moist chamber.
10. The slide was washed twice in 2XSSCN for 4 minutes at 37 刟C.
11. The slide was incubated with rabbit anti-mouse FITC conjugated secondary antibody [Zymed 62-6411] (37.5僘磄/ml) for 30 minutes at 37 刟C in a moist chamber.
12. The slide was washed twice in 2XSSCN for 4 minutes at 37 刟C.
13. Counterstaining of the slide was done with propidium iodide (2僘磄/ml) [Sigma P-4170] for 50 seconds, followed by rinsing in water and embedding in DABCO [Sigma D-2522] anti-fade solution.
14. The slide was visualized under a Zeiss fluorescence microscope using appropriate filters.
15. Photography was done using Kodak 400ASA film, 120s exposure and aperture setting automatic.

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Chromosome preparation and Fluorescence in-situ hybridizati