Fluorescent in situ hybridization.

REQUIREMENTS
Trude Schwarzacher ts32@le.ac.uk
Department of Biology, University of Leicester, UK
http:www.molcyt.com
This practical is run in two parts. Part A takes place on the afternoon of the first
day and involves setting up the experiment. Part B takes place on the following
day and involves stringent washing and detection of hybridization signal
We should divide participants into groups, best would be groups of 2, but we can also work with
groups of 3 or 4 aiming for 4-6 groups in total. It depends on space in incubators, humid chambers
and coplin jars we have, and how many slides we are getting together; but each student should have
at least 1 slide (better 2 slides) and each group at least one staining cuvette/coplin jar which holds 68 slides (probably 2x banana, 2x triticale and 2x 1B/1R wheat and maybe a ginger).

Equipment and consumables per group of 2-4 students

1x P2 pipetman
1x P20 pipetman
1x P 200 pipetman
1x P1000 pipetman
1x box sterile P2 pipette tips
1x box sterile P20/P200 pipette tips
1x box sterile P1000 pipette tips
20x 1.5ml eppendorf tubes
1x eppendorf rack
1x tissues or blue roll
1x Floaty
1x waste pot
1x ice bucket + ice
1x Markepen
1x Scissors
1x fine forcep
1x ordinary forcep
1x 250ml measuring cylinder
1x 100ml measuring cylinder
2x 500ml duran bottles
1x Timer
Plastic hazard bag (cloudy ones) cut A5 but left double
1x Plastic rack to dry glass slides (for 15ml centrifuge tubes, but less high such
that glass slides fit diagonally into slots)
1x tall plastic coplin jar (staining cuvette) for 8 slides with lids
24 x 40 coverslips size 0 (15 per group or one box for all)
Filterpaper (9cm disk or squares)
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o

Equipment, material and consumables to have in the lab and to share between groups
Equipment
o
humidity chambers (tins), best would be one per group, but we probably can
share depending on size of chamber
o
Pair of rods for humidity chambers
o
Thermometer (at least one, but better one per group)
o vortex mixer
o 37oC incubator or waterbath
o 1x or 2x Waterbath with variable temperature between 45oand 80o
o
microcentrifuge for 1.5ml eppendorfs (at least one, but better one per group)
o 1x or 2x Platform shakers
Solutions
o
Aliquots of 2mls high grade distilled water
o Tween-20
o 20 x SSC in 5 litre container
o 2x SSC in 5 litre container
o Distilled water in 5 litre container
o 2x Icebuckets with ice for special chemicals supplied by operator
Part A
o 2x Thermocycler or heat blocks for in-situ hybridization
o
Master mix for setting up FISH to be put in ice bucket
o
Labelled probes to be put in ice bucket
Part B
o DAPI
o Antifade

Preparation Notes
Chemicals
o 20X SSC
o 2X SSC
o 200ml 70% ethanol
o 200ml 80% etanol
o 200ml 96 or 100% ethanol
Mastermix
One aliquot of size and concentration below is required for each group of 2-3 students, Prepare in
1.5 ml microcentrifuge tubes unless otherwise stated. Labelled with underline descriptor.
FOR PART A:
1. Mastermix:
Component

Final
concentration in
hybridization mix

Amount for
1 slide

6 SLIDES
(Multiplied by 7)

4 SLIDES
(Multiplied by 5)

50%

20 l

140l

100l

2x

4 l

28l

20l

10%

8 l

56l

40l

Salmon sperm DNA 1g/l

0.025g

1 l

7l

5l

100mM EDTA
10% SDS

1.25 mM

0.5l

3.5l

2.5l

0.125%

0.5l

3.5l

2.5l

34l

238l

170l

100% Formamide
20x SSC
50% Dextran sulphate

Total

See Protocol (appendix A3) for how to prepare components.

Heat Dextran sulphate before use and cut of tip of dispensing tip;
Mix well than freeze. Can be prepared several weeks before practical
2. Labelled probes
From Operator: 45S rDNA, 5s rDNA and genomic rye DNA
3. Slides
From Operator; these should be pre-treated as under 4. Either on Monday before the course or on
Tuesday during the chromosome preparation session.
From Particpants; these we will just use as they are without pre-treatment
4. Pre-treatment of Operator plant chromosome slides (see schedule below):

There will be 12-16 slides to be treated in 2 coplin jars, so prepare 200ml 4% formaldehyde; no
pepsin treatment needed.
FOR PART B:
5. DAPI
500 l aliquots of 4g/ml (stock solution in freezer in lab or from Lynne)

DAPI (4',6-diamidino-2-phenylindole, Sigma). Prepare DAPI stock solution of 100 g/ml in

water. DAPI is a potential carcinogen. To avoid weighing out the powder, order small quantities
and use the whole vial to make the stock solution. Aliquot and store at 20C (it is stable for
years). Prepare a working solution of 4 g/ml by dilution in McIlvaine's buffer, aliquot and store
at 20 C.
6. Antifade
Use ours (Citifluor AF1, Agar) or the one supplied by Lynne
500 l aliquots

PRETREATMENTS
Air dried slides should be kept at room temperature at least overnight before use, for
longer periods store with silica gel at -20C.
Post-fixation of air dried slides:
ethanol/acetic acid 3:1 for 10-30min.
100% ethanol for 5 min.
100% ethanol for 5 min.
air-dry.
Rnase treatment:
1. 1.Make 100g/ml Rnase solution by diluting Dnase-free Rnase stock in 2xSSC allow
250l per slide.
2. [10mg/ml stock 1:100 with 2xSSC or 1mg/ml stock 1:10 with 2xSSC]
3. Apply 200l Rnase solution to each slide, cover with a plastic cover slip.
4. Incubate for 1h at 37C in humid chamber.
5. Wash in 2xSSC for 2 min.
6. Wash in 2xSSC for 10 min
Pepsin treatment not needed
Paraformaldehyde fixation:
7. Start to prepare paraformaldehyde before putting slides in Rnase: In the fume hood add
4g paraformaldehyde to 80ml water. Heat to 60C for 10 min, add a few drops of 4M NaOH
to clear the solution. Cool down to room temperature. Adjust pH to 7 with H2SO4. Make up
to final volume of 100ml with water. N.B. One drop of NaOH leaves the solution at
approximately the correct pH.
8. In the hood, incubate slides in paraformaldehyde at RT for 10 min.
9. Wash in 2xSSC for 2min.
10. Wash in 2xSSC for 10min
Dehydration:
11. Wash in 70% ethanol for 2 min. (discard after use)
12. Wash in 85% ethanol for 2 min. (keep in a storage to prepare 70% ethanol)

13. Wash in 100% ethanol for 2 min. (keep in a storage bottle for reuse; to prepare 70%, 85%
ethanol)
14. Air-dry.