Comunidad

The FISH experiments on Dynamic Molecular Combed (combed) DNA involved 5 steps:

i Slide preparation

ii Probe labelling and preparation iii Hybridisation

iv Post-hybridisation washes V Detection and amplification

2.2.14.1 Slide preparation

The slides had previously been "combed" according to the method of (Michalet et al, 1997) and stored at -20°C, they were allowed to warm to room temperature and then dehydrated through an Ethanol series for 3 minutes in each of 70%, 90% and 100% ethanol solutions. The slides were then denatured by incubation for exactly 2 minutes in 70% formamide/2xSSC at 75°C, and then transferred into a solution of cold 70%

ethanol (stored in a coplin jar at -20°C) for 3 minutes. The slides were then dehydrated again by passing through a 90% and 100% ethanol series for 3 minutes at each step. The slides were then allowed to dry in a 37° incubator.

2.2.14.2 Probe labelling and preparation

The DNA probes to be used were labelled with Biotin or Digoxigenin depending on the design of the experiment performed.

2.2.14.2.a Biotin labelling

Biotin labelling was performed using the BioNick kit from Gibco BRL (No 18247-015). One microgram of template DNA was added to lOjil of lOx dNTP mix, lOpl of enzyme mix, both from the kit, and made up to a volume of 90p,l with sterile double distilled water. The mixture was spun down briefly and then incubated for 90 minutes at 16°C, the reaction was stopped by adding 5|li1 of stop buffer, supplied with the kit. The labelled probes were stored at -20°C until needed.

2.2.14.2.b Digoxigenin labelling

Digoxigenin labelling was performed using the Nick Translation kit from Boehringer (No 976776). One microgram of template DNA was added to 20^1 of lOx dNTP mix (made by adding the following volumes of 0.4M stock solutions 6|Li1 - dATP, 6|il - dCTP, 6|il dGTP, 4|Li1 - dTTP and 2|li1 of Dig-11-dUTP (Boehringer)), 4|Li1 of enzyme mix, and made up to a volume of 40pl with sterile double distilled water. The mixture was spun down briefly and then incubated for 90 minutes at 16°C, the reaction was stopped by adding 2|xl of 0.5M EDTA (pHS.O). The labelled probes were stored at

-20°C until needed.

Approximately 200-500ng of labelled probes, per slide, were mixed with a 5 times excess of Cot-1 DNA (Gibco), 20|Lig of Herring or Salmon sperm DNA (Sigma), 1/10th volume of 3M Sodium Acetate and at least two volumes of cold 100% Ethanol. The solution was allowed to precipitate for 30-45 minutes at -70°C and then spun in a microcentrifuge at 13000g for 10 minutes at room temperature. The supernatant was discarded and the pellet dried in a vacuum dryer for 10 minutes. The DNA pellet was resuspended in lOpl hybridisation solution for FISH (see appendix 1) and warmed to 45°C for 15 minutes to aid resuspension, the DNA was then denatured in boiling water for 5 minutes and then snap cooled by placing on ice. The tubes with the denatured probe DNA were briefly spun down prior to use.

2.2.14.3 Hybridisation

The labelled and denatured probe was pipetted onto a 22 x 22mm coverslip and picked up by inverting the slide with the template DNA over the coverslip until the slide and coverslip stick together. The edges of the coverslip were sealed with Cow gum and the slides incubated at 37°C for, at least, 16 hours or more.

2.2.14.4 Post-hybridisation washes

After the hybridisation, the Cow gum and the coverslip were carefully removed from the slides and the slides were washed in 50% formamide/2xSSC at room temp, three times for 5 minutes each. The slides were then washed three times in 2xSSC at room

temperature for 5 minutes each. All the washes were performed in a coplin jar on a shaker at -lOOrpm. After the washes each slide had 50|ll1 of blocking solution (see

appendix 1) added, on a coverslip, and were incubated in a moist chamber at 37°C for 30 minutes.

2.2.14.5 Detection and amplification

The detection protocol used 5 layers of antibody-fluorochrome to amplify the signal. The slides were subjected to 5 cycles of detection and washing each detection step using the appropriate reagent from Table 2.2.4. The detection steps were incubated for 30 minutes at 37°C in a moist chamber and each washing step involved 3 washes in 4xSSC/0.05% Tween-20 (see appendix 1) for 5 minutes in a coplin jar on a shaker at -lOOrpm, the coplin jar was covered by a larger beaker which was covered in tin foil to block out light. After the last layer of amplification has been added the slides were rinsed in Ix PBS for 5 minutes in a coplin jar on a shaker at -lOOrpm and then allowed to drain on a tissue before a drop of Vectashield (Cambio) was placed on the slide and covered with a 22 x 22mm coverslip. The excess Vectashield was blotted off by pressing the slide between two tissues, the coverslip was then sealed down with clear nail polish. The slides were stored in the dark at all times and could be kept at 4°C for a few weeks without losing signal strength.

Table 2.2.4 Signal amplification for FISH experiments

layer Biotin amplification dilution Digoxigenin amplification

dilution

1 Avidin-Texas red* 1/50 mouse anti-Dig FITC^ 1/50 2 Biotinylated anti Avidin* 1/100 donkey anti-mouse FITC^ 1/50 3 Avidin-Texas red* 1/50 mouse anti-rabbit FITC^ 1/50 4 Biotinylated anti Avidin* 1/100 anti FITC (Fl)^ 1/400 5 Avidin-Texas red* 1/50 anti anti FITC (F2)^ 1/100

* = Vector Laboratories A = Interchim - France t= Cambio