DEPARTMENT OF BIOLOGY

Fluorescent in situ hybridization.REQUIREMENTS Trude Schwarzacher ts32@le.ac.ukDepartment of Biology, University of Leicester, UKhttp: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 6-8 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)

Equipment, material and consumables to have in the lab and to share between groups

Equipment humidity chambers (tins), best would be one per group, but we probably can share depending on size of chamber Pair of rods for humidity chambers Thermometer (at least one, but better one per group) vortex mixer 37oC incubator or waterbath 1x or 2x Waterbath with variable temperature between 45oand 80o microcentrifuge for 1.5ml eppendorfs (at least one, but better one per group) 1x or 2x Platform shakers

Solutions Aliquots of 2mls high grade distilled water Tween-20 20 x SSC in 5 litre container 2x SSC in 5 litre container Distilled water in 5 litre container 2x Icebuckets with ice for special chemicals supplied by operator

Part A 2x Thermocycler or heat blocks for in-situ hybridization Master mix for setting up FISH to be put in ice bucket Labelled probes to be put in ice bucket

Part B DAPI Antifade

Preparation Notes

Chemicals 20X SSC 2X SSC 200ml 70% ethanol 200ml 80% etanol 200ml 96 or 100% ethanol

MastermixOne 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:

ComponentFinal concentration in hybridization mixAmount for 1 slide6 SLIDES(Multiplied by 7)4 SLIDES(Multiplied by 5)

100% Formamide50%20 l140l100l

20x SSC2x4 l28l20l

50% Dextran sulphate10%8 l56l40l

Salmon sperm DNA 1g/l0.025g1 l7l5l

100mM EDTA1.25 mM0.5l3.5l2.5l

10% SDS0.125%0.5l3.5l2.5l

Total34l238l170l

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 probesFrom Operator: 45S rDNA, 5s rDNA and genomic rye DNA

3. SlidesFrom 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. DAPI500 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 100g/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 4g/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 Lynne500 l aliquots

PRETREATMENTSAir 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 minPepsin 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.