Fluorescence In Situ Hybridisation Protocol - This product is for research use only
Introduction
The FISH protocol is divided into two stages. Denaturation and Hybridisation are performed on day one. Washing and Detection are performed on day two. The metaphase spreads are normally prepared a day in advance. On day one, the DNA of the chromosomes and paints is denatured and the Hybridisation process (reannealing) takes place overnight. On day two the slides are washed to remove unbound DNA sequences followed by detection, counterstaining and mounting.
A PDF version of this protocol can be found here:- Hybridisation Protocol
Denaturation and Hybridisation
Approx. time:
Slide preparation: 30 min + overnight (or + 90 min).
Denaturation and hybridisation: 30 min + overnight.
Note: Metaphase spreads on slides can be prepared on previous day and aged overnight at room temperature, or on day one and aged for 90 min on hot plate.
| Requirements (not provided) | |
|---|---|
| Equipment | Reagents |
| Ethanol-cleaned slides | NaCl |
| Coverslips | HCl |
| Eppendorf tubes | Na citrate |
| Coplin jars | Double distilled water |
| Humidified chamber | Pepsin (SIGMA, p7000) |
| Micro-pipette 1µl, 10µl, 500µl | Formamide (BDH, 103264R) |
| Pipette 10ml, 20ml | Absolute Ethanol |
| Vortex | Fixogum rubber cement |
| Parafilm | Clear nail varnish |
| Micro-centrifuge | |
| 45°C Water bath | |
| 37°C Incubator | |
| Fluorescence microscope with a suitable filter set | |
| Solutions to be prepared: | |
|---|---|
| 20X SSC | |
| 2X SSC | |
| Denaturation solution | |
| Stringency wash solution | |
| Pepsin solution (optional) | |
| Solution: 20X SSC | |
|---|---|
| 87.6g NaCl | |
| + | 44.1g Na citrate |
| Make up to 500ml in double distilled water | |
| Adjust pH to 7.4 using concentrated HCl (before finalizing water volume). Aliquot and autoclave | |
| Solution: 2X SSC | |
|---|---|
| 50ml 20X SSC | |
| + | 450ml Double distilled water |
| 500ml 2X SSC. Mix well | |
| Denaturation solution | |
|---|---|
| 70ml Deionised formamide | |
| + | 30ml 2XSSC |
| 100ml Denaturation Solution. Mix wel | |
| Pepsin solution - (optional) | |
|---|---|
| 0.5ml Stock pepsin solution (1% in water) | |
| + | 49.5ml 10mM HCl |
| 50ml Pepsin solution | |
| Note: Stock solution can be stored at -20°C in small aliquots. | |
Note: All volumes of the working reagents are for ten slides.
Procedure:
Pepsin treatment (optional):
Slides can be treated on previous day:
- Drop metaphase onto clean slide and dry at room temperature.
- Check slide with phase contrast microscope.
- Dehydrate slide in 100% ethanol for 5 min and dry at room temperature.
- Incubate slide in pepsin solution for 2-5 min (depending on amount of cytoplasm).
- Wash in 2XSSC for 1 min. Repeat twice and rinse briefly in distilled water.
- Dehydrate by serial ethanol washing for 2 min each in 70% (v/v) ethanol,70%, 90%, 90%, and 5 min 100%. Dry at room temperature.
Note: Pepsin treatment replace steps 3 and 4 of slide preparation and denaturation
Probe preparation/denaturation
- Warm chromosome paints to 37°C, vortex and centrifuge for 1-3 seconds.
Note:There are two kinds of paints:- Ready to use paint use 15µl per slide.
- Concentrated paint (Human, Mouse) add 3µl of chromosome paint to 12µl of hybridisation buffer.
- For dual colour painting, add 3µl of each concentrated paint to 9µl of hybridisation buffer.
Use a total of 15µl for a 34X22mm slide, 10µl for a 22X22mm slide.
- Denature probe for 10 min at 65°C, and hold at 37°C for 30-60 min.
Slide preparation/denaturation
- Prepare new slides with fresh metaphase spreads, which have been fixed with 3:1 methanol: acetic acid.
- Dehydrate by serial ethanol washing for 2 min each in 70% (v/v) ethanol, 70%, 90%, 90%, and 5 min 100%. Age for 60 min at 65°C.
- Denature slide by incubating in pre-warmed Denaturation solution at 65°C for 1.5-2 min.
- Quench slides in ice-cold 70% (v/v) ethanol for 4 min and dehydrate by serial ethanol washing for 2 min each in 70% (v/v) ethanol, 70%, 90%, 90%, and 5 min 100%. Dry at room temperature.
Note: Denaturation of the slides is an important step. Be sure that Denaturation solution is at the right temperature. Some slides benefit from 1.5 min denaturation and others up to 2 min. The right timing, which is determined by trial and error, depends on type of cells used, metaphase preparation, brand of formamide, etc.
Hybridisation
- Apply probe (15µl*) onto the slide. Apply coverslip and remove air bubbles by gently pushing on coverslip with a pencil. Seal with rubber cement.
- Place slide in an air tight, prewarmed humidified chamber and incubate overnight in the dark at 37°C.
Note: With good quality metaphase spreads a minimum period of 4 hrs can be sufficient for hybridisation.
Post hybridisation washes are incorporated in detection protocol as part of day two.
