Detection Protocol - 1089-KB

Biotin labelled chromosome detected with FITC

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:- Biotin Detection - 1089-KB Protocol

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

Requirements (not provided)
Equipment	Reagents
Coverslips	NaCl
Eppendorf tubes	Na citrate
Coplin jars	Double distilled water
Humidified chamber	HCl
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
1X SSC
4X SSC
Detergent wash solution
Stringency wash solution
Working Reagent A
Working Reagent B

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: 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

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

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

Working Reagent A:
	190µl Blocking Protein BP
+	1060µl Detergent wash solution
	1250µl Diluted Blocking Protein (15%)

Working Reagent B:
	2.5µl Detection reagent Y1
+	1247.5µl Working Reagent A
	1250µl Diluted Detection reagent Y1 (1:500)
Incubate in the dark for 5 min. Microcentrifuge at 11.000g for 5 min.

Procedure:

Washing

1. Pre-warm solutions to 45°C in water bath at least 30 min before starting:
   • Two Coplin jars of Stringency wash solution (50ml each)
   • Two Coplin jars of Solution 1XSSC (50ml each)
   • One Coplin jar of Detergent wash solution (50ml)
   
   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 1XSSC for 5 min. Take off rubber cement and replace in Solution 1XSSC to remove the coverslip.
   Note: Do not allow the slide to dry.

   Stringency washes:

   • Wash slides twice by incubating 5 min each time in Stringency wash solution (45°C).
   • Wash slides twice by incubating 5 min each time in Solution 1XSSC (45°C).
   • Incubate slide 3 times for 4 min in Detergent wash solution (45°C).

   Detection

3. Apply 100µl of Working Reagent B onto the slide and cover with Parafilm immediately.
4. Incubate slide in a humidified chamber for 15-20min at 37°C.
5. Remove Parafilm from the slide and wash 3 times for 4min each time in the Detergent wash solution at room temperature.
6. Drain slide well and mount with 25-50µl of Reagent MD (25µl for 22x22 coverslip; 50µl for 22x50 coverslip).
7. Apply glass coverslip and seal with nail varnish. Store slides in the dark at 4°C.
   Note: You get almost no air bubbles when supplied Reagent MD is applied on the coverslip and the almost dry (but not dried out!) slide is laid face-down on the coverslip.
8. View slides using standard epifluorescence filters for FITC and for counterstain DAPI.

Troubleshooting

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