Thursday, October 16, 2003

Description

染色体准备和Fluorescence in-situ hybridization (FISH) 方法

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.