Fluorescence in situ hybridisation (FISH )

Fluorescence in situ hybridisation was performed using standard methods (Baldini and Lindsay, 1 9 9 4 ).

2.2.22.1 Preparation of metaphase spreads

A 50 ml culture of lymphoblastoid cells was prepared and the media changed 2 4 hours prior to preparation of m etaphase spreads. After 2 4 hours the cells w ere mitotically arrested by the addition of 250pl of lOpg/ml colcemid and incubation for 1 hour. The

cells were pelleted by centrifugation at| 1 6 0 g for 5 minutes, and the media

aspirated. Cells were resuspended in the remaining media and 50ml of freshly

prepared 75m M KOI hypotonic solution was added dropwise. Cells were spun down and resuspended in the remaining supernatant. Ice cold fix (3:1 methanol: glacial acetic acid, freshly prepared) was added dropwise, and the volume made up to 10 ml. Cells were mixed by inversion and incubated on ice for 20 min. Cells were pelleted by centrifugation at 1500 rpm for 5 minutes and resuspended dropwise in a further 10 ml of fresh fix. The cells were fixed twice more and dropped onto slides, previously cleaned with ethanol. Slides were stored at 4°C with desiccant until required.

Metaphase chromosomes in fix were kept at 4°C for short term storage and at -20°C for longer term storage.

2.2.22.2 Nick translation of DNA probes

Nick translation was carried out using the BioNick kit from BRL. Cosmid mini-prep DNA was prepared as described in section 2.2.2.S. Prior to use as template DNA, lOpg/ml of RNase A was added, and the mix incubated at 37°C for 1 hour. The DNA was then extracted and precipitated as described in section 2.2.2. The nick translation was set up as follows: Ip g of DNA was mixed with 5pl of lOx dN TP mix, 5|il of the lOx enzyme mix, and the volume increased to 50|il. The reaction was incubated at 12-16°C for 2 hours. The reaction was stopped by the addition of 4pl of 0.5M EDTA and lp.1 of

10% (w/v) SD S. The probe was separated from unincorporated nucleotides by spinning through a G -50 Sephadex column, equilibrated in 50 mM Tris-CI, 1 mM EDTA, 0.1% (w /v) SD S at 3 5 5 g for 5 minutes. A lOpI aliquot of the probe was electrophoresed in a 2% (w/v) agarose gel to estimate the fragment size and quantity.

The probe was kept at -20°C until required.

2.2.22.3 Hybridisation

Prior to use metaphase slides w ere aged in 2x SS C at 37°C for 30 min and dehydrated through 70% (v/v), 90% (v/v) and absolute ethanol for 3 min each and air dried. Slides w ere denatured by immersion in 70% (v/v) deionised formamide, 2x S S C

equilibrated at 70°C , for 2 minutes exactly. The slides were then dehydrated by dipping successively in 70% (v/v) ethanol (prechilled at -2 0 °C ), 90% ( v/v) ethanol and absolute ethanol. The slides w ere left to air-dry at an incline.

The probes w ere repetitive so it was necessary to compete them with competitor DNA to suppress background prior to the hybridisation. A lOpI aliquot of labelled probe was mixed with 2pl of C^t 1 human competitor DNA and 3pl of salmon sperm DNA

pH5.2 and 60pl of ethanol. It was incubated on dry ice for 15 minutes or at -70°C for 30 minutes, then pelleted by centrifugation at 14 000 rpm at 4°C for 20 minutes. The pellet was allowed to air-dry, then resuspended in lOpI of the hybridisation solution. The probe was denatured by incubation at 80°C for 5 minutes, followed by annealing at 37°C for 15 minutes, after which it was placed on ice until required.

The slides w ere prewarmed, then lOpI of the competed probe in the hybridisation solution was pipetted over the selected area , and a coverslip ( 2 2 x 2 2 mm) was gently

lowered onto the solution, being careful not to trap any air bubbles. The rim of the coverslip was sealed with Cow Gum rubber cement. The slides were transferred to a moist box, and incubated horizontally overnight at 37°C .

2.2.22.4 Visualisation

Post hybridisation, the slides w ere washed three times in 50% (v/v) formamide, 2x SSC at 4 2°C for 5 minutes, followed by three washes in 1x SSC at 6 0°C for 5 minutes. To decrease non-specific hybridisation, 200|il of blocking buffer was applied to each slide, covered with a 50 x 2 2mm coverslip, and incubated at 3 7°C for 30 minutes. The

rest of the procedure was carried out in the minimum of light to protect the

fluorochromes. The antibodies were then applied in a similar manner diluted in FITC buffer to a final concentration of 7ng/|il, and incubated at 37°C for 30 minutes to 1 hour. This was followed by three successive washes in 4 x SSC , 0.1% (v/v) Tw een 20 at 42°C for 5 minutes each. The chromosomes were stained by immersing the slides in DARI (200ng/m l), 2x SS C for 10 minutes at room tem perature. The slides w ere then mounted using Vectashield mountant from Vector Labs, Inc.

2.2.22.5 Fluorescence microscopy

The slides were examined in diminished lighting on a Ziess Axioscope 20 fluorescence microscope under oil using a lOOx objective. A positive result was confirmed after similar results were obtained using more than one probe (if possible) and upon examination of several different metaphase spreads. Images were photographed using a Photometries Nu200 C C D cam era system and manipulated using the SmartCapture software from Digital Scientific. Images were printed from a M acDraw Pro format on a M itsubishi sublim ation printer.