Fluorescence in situ Hybridisation Protocol - HFISH

HarlequinFISH

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:- HarlequinFISH Protocol

Denaturation and Hybridisation   (This product is for research use only)

Approx time:

Slide Preparation: 90 min
Denaturation and hybridisation: 30 min + 2 days

Requirements (not provided)
Equipment	Reagents
Non fluorescent slides	NaCl
Non fluorescent Coverslips	Na citrate
Eppendorf tubes	HCl
Coplin jars	pepsin (SIGMA, P7000)
Humidified chamber	Formamide (BDH, 103264R)
Micro-pipette 1µl, 10µl, 500µl	Absolute Ethanol
Pipette 10ml, 20ml	Double distilled water
Vortex	Fixogum rubber cement
Parafilm	Clear nail varnish
Micro-centrifuge	DAPI II®
45°C Water bath
37°C Incubator
Fluorescence microscope with a suitable filter set

Solutions to be prepared:
20X SSC
2X SSC
Denaturation 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

Solution: Denaturation solution
	70ml Foramide
+	30ml 2X SSC
	100ml Denaturation solution. Mix well

Solution: Pepsin solution
	500µl Stock pepsin soln (1% in water)
+	49.5ml 10mM HCI
	50ml Pepsin solution
Note: pepsin stock solution can be stored at -20°C in small aliquots

Procedure

Pepsin treatment (recommended):

Slides can be pepsin treated on the previous day:

• 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 50ml 2X SSC for 5 min. Repeat and then 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 ethanol%.
• Dry at 65°C for 1 hour.
  Note: Pepsin treatment replaces step 3 to 8 of Slide preparation and denaturation.

  Probe preparation and denaturation:

1. Warm up the HarlequinFISH paints in a 65°C waterbath for 5 min. For each hybridisation, aliquot 10µl of probe into a 500µl Eppendorf tube.
2. Denature at 65°C - 72°C for 10 min and then allow the probes to preanneal at 37°C for 10 - 60 min. (The denaturation of probe can be either performed in a waterbath, or ideally in a PCR machine).

   Slide preparation and denaturation:

3. Prepare 4ml of fresh 3:1 fixative (methanol: glacial acetic acid).
4. Place tube of metaphase suspension on ice.
5. Take slides out which have been pre-cleaned and stored in 100% ethanol, wipe off ethanol on the surface with tissue paper and air-dry. Place on a horizontal surface.
6. Resuspend the cells in the fixative by flicking tube gently. Place a single drop of cell suspension onto a slide. Check the spreading of chromosomes under phase contrast microscope. If under-spread, add a drop of fresh fixative immediately after the cell suspension is placed on the slides. Please make sure that the metaphase spreads are free of cytoplasm. Leave slide to dry in the air.
7. Dehydrate by serial ethanol washing for 2 min each in 70% (v/v) ethanol, 70%, 90%, 90%, and 5 min 100% ethanol. Dry at room temperature.
   Note: If cytoplasm is still present on the slides treat the slides with pepsin (see recommended pepsin treatment)
8. Age for 90 min on 65µC hot plate.
   Note: If you have pre treated the slides a day in advance and age them overnight at room temperature you do not need to age for 90 min on 65µC hot plate.
9. Denature slide by incubating in pre-warmed Denaturation solution at 70µC for 1.5-2 min.
10. 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:

11. Apply probe (10µl) onto the slide. Apply coverslip and remove air bubbles by gently pushing on coverslip with a pencil. Seal with rubber cement.
12. Place slide in an air tight, prewarmed humidified chamber and incubate in the dark at 37°C for 36-48 hrs.

Washing & Detection (day 2)

Approx time: Preparation 20 min + Procedure 74 min

Solutions to be prepared:
1X SSC
4X SSC
Detergent wash solution
Stringency wash solution
Solutions to be made:
Diluted Blocking Protein
Diluted Detection Reagents

Solution: 1X SSC
	25ml 20X SSC
+	475ml Double distilled water
	500ml 1X SSC. Mix well

Solution: 4X SSC
	100ml 20X SSC
+	400ml Double distilled water
	500ml 4X SSC. Mix well

Solution: Detergent wash solution
	500ml 4X SSC
+	250µl Detergent DT
	500ml Detergent wash solution. Mix well

Solution: Stringency wash solution
	50ml Formamide
+	50ml 1X SSC
	100ml Stringency wash solution. Mix well
Note: Stringency wash solution can be reused up to 5 times but should be discarded after 2 months

Solution: Diluted Blocking Protein
	95µl Blocking Protein BP
+	530µl Detergent wash solution
	625µ Diluted Blocking protein (15%)

Solution: Diluted Detection reagents (DDR)
DDR Y2 (Cy5)+F1 (FITC)	0.25µl Y2
	0.625µl F1
+	125µl Detergent wash solution
	125µl Diluted Detection reagent Y2+F1
DDR F2 (FITC)	0.625µl F2
+	125µl Detergent wash solution
	125µl Diluted Detection reagent F2 (1:200)
Note: All volumes of these working solutions are for five slides.

Procedure

Washing:

1. Pre-warm solutions to 45°C in a water bath at least 30 min before starting:
   • Two coplin jars of 1X SSC
   • Two coplin jars of 50% formamide/1X SSC (V/V)
   • One coplin jar of Detergent Wash solution
   
   Note: The temperature is important. Check the temperature of the solutions in the Coplin jar and not of the water in the water bath.
2. Take out the slide from the incubator and leave in Solution 1X SSC for 5 min. Take off rubber cement and leave in Solution 1X SSC to remove the coverslip.
   Note: Do not allow the slide to dry.

   Stringency washes:

   • Wash slides twice by incubating 5 min in each Stringency wash solution (45°C).
   • Wash slides twice by incubating 5 min in each Solution 1X SSC (45°C).
   • Incubate slide 3 times for 4 min each time in Detergent wash solution. (45°C) by emptying and refilling the Coplin jar.

   Optional Blocking protein step - if omitting go to step 19

3. Apply 125µl of diluted blocking protein onto the slide and cover with Parafilm immediately.
4. Incubate slide in a humidified chamber for 15-20 min at 37°C.
5. Remove Parafilm from the slide and wash 3 times 4 min each in Detergent wash solution at room temperature by emptying and refilling the Coplin jar.
6. Apply 125µl pre-combined Detection reagent Y2 and F1 onto the slide and cover with Parafilm immediately.
7. Incubate slide in a humidified chamber for 15-20 min at 37°C.
8. Remove Parafilm from the slide and wash 3 times 4 min each in Detergent wash solution at room temperature by emptying and refilling the Coplin jar.
9. Apply 125µl Diluted Detection Reagent F2. Cover with parafilm immediately and incubate at 37°C for 15-20 min.
10. Remove Parafilm from the slide and wash 3 times 4 min each in Detergent wash solution at room temperature by emptying and refilling the Coplin jar.
11. Drain slide well and mount with 50µl of DAPI II®.
12. Apply glass coverslip and seal with nail varnish. Store slides in the dark at 4°C.
    Note: You get almost no air bubbles when DAPI II® is applied on the coverslip and the almost dry (but not dried out!) slide is laid down on the coverslip.
13. View slides using epifluorescence filters specific for Cy3, Cy5, and FITC.

Troubleshooting

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