Chromosome preparation and Fluorescence in-situ hybridizati

Thursday, October 16, 2003

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

Procedure
Y 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 aC 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 aC for 1 week for ageing.

FISH

1. Normal human chromosome spreads were prepared according to standard cytogenetic methodology .
2. Slides were treated with RNase (100Yg/ml) [Sigma R-9009] for 45 minutes at 37 aC 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 aC.
4. Slides were passed through an ethanol series (70%, 85% and 100%) for 3 minutes each and stored at V20 aC till use.
5. 20ng of labeled probe DNA was denatured in a solution containing 13Yl formamide (68%) [Sigma F-7503], 1Yl of 2X SSC (0.1X), 3Yl of 10% dextran sulphate (1.6%) and 1Yl of 10Yg/Yl calf thymus DNA (0.52Yg/Yl) [Sigma D-4522] for 5 minutes at 70 aC. Slides containing target chromosomal DNA were denatured in 70% formamide [Sigma F-7503] and 2X SSC for 8 minutes at 70 aC 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 aC in a moist chamber.
7. Stringency washing was done in 50% formamide [Sigma F-7503] and 2X SSC for 10 minutes at 37 aC; 2X SSC thrice for 4 minutes each at 37 aC; and in 2X SSC with 0.1% Nonidet P40 (ethylphenylpolyethyleneglycol) [Fluka 74385] for 5 minutes at 37 aC.
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] (40Yg/ml) for 45 minutes at 37 aC in a moist chamber.
10. The slide was washed twice in 2XSSCN for 4 minutes at 37 aC.
11. The slide was incubated with rabbit anti-mouse FITC conjugated secondary antibody [Zymed 62-6411] (37.5Yg/ml) for 30 minutes at 37 aC in a moist chamber.
12. The slide was washed twice in 2XSSCN for 4 minutes at 37 aC.
13. Counterstaining of the slide was done with propidium iodide (2Yg/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