In situ hybridization - a 'kit' for a course

 

The following list of things is needed to demonstrate in situ hybridization

As we all share this process and rely on each other please share the following:

  1. No 5 fine forceps

  2. Fine curved forceps

  3. Plastic forceps

  4. Syringe needles

  5. Single edge razor blades

  6. 18x18 coverslips

  7. 22x40 coverslips

  8. Cleaned microscope slides

  9. Diamond pencil

  10. Black tiles

  11. Autoclavable plastic bag

  12. Digital thermometer

  13. Coplin jars (2)

  14. USB memory stick disk (or media for camera: CF, MemoryStickDuo, 400 ASA colour print film

  15. Blank CD ROMS

  16. Clock Timers

  17. 20x SSC (2ml and 50ml)

  18. Formamide (2ml and 40ml)

  19. Tween 20 detergent (2ml)

  20. Enzyme mixture (5ml)

  21. Enzyme buffer (10x; 15ml)

  22. Ethanol (50ml)

  23. Acetic acid (50ml)

  24. Plastic Pasteur pipettes

  25. Gilson 20ul and tips

  26. Whatman 100mm filter paper circles

  27. Lens tissue

  28. Hand lens

  29. Torch for microscope room

  30. Marker pens

  31. Post-it markers

  32. Glasses

1ml each of following:

  • 10% SDS

  • 50% dextran sulphate

  • 100 mM EDTA

  • Molecular biology grade water

  • Salmon sperm DNA

  • RNase

  • Pepsin

  • Fluorescence immersion oil

  • DAPI 4ug/ml

  • PI Propidium iodide

  • CMA Chromomycin A3

  • Mounting medium (Citiflour, VectorShield)

Probes: preferably direct-fluorochrome labels

  • 5S rDNA

  • 45S rDNA

  • Telomere, mammalian (TTAGGG)7 and plant (TTTAGGG)7

  • SSRs

  • Genomic rye DNA

Fixations and prepared slides

  • In situ slides already DAPI stained

Seed germination:

  • 90 mm Petri dishes

  • Rye , wheat, 1B/1R wheat, Brassica, Arabidopsis

  • Fixations as appropriate

Chemicals and solutions Trude Schwarzacher Water:
For molecular biology (labelling.labeling reactions, solutions of DNA, hybridization mixtures etc): aliquots of purchased molecular biology grade water.
For seed germination: purchased bottles of drinking water (this is free of chlorine, metals from pipes, dead rats from the roof water tank, etc.). 

WE RECOMMEND THE USE OF THESE TYPES OF WATER STRONGLY: MANY EXPERIMENTS FAIL THROUGH USING THE WRONG WATER.
For stock solutions and washing buffers we use autoclaved deionised water from a standard laboratory system.

500mM EDTA pH 8 Dissolve 186.1 g disodium ethylene diamine tetraacetate.2H 2 O in 800 ml of water. Add about 10 g NaOH pellets, stir on a magnetic stirrer and adjust to pH 8 with further NaOH (about 10 g pellets). Make up to 1 l and sterilize by autoclaving.

  • We will need 10ml

  • Make 2ml of a 100mM solution made with pure water

20x SSC (saline sodium citrate, salt sodium citrate or standard saline citrate)

  • 3M sodium chloride

  • 300mM trisodium citrate

  • Make 1 litre and autoclave.

  • Prepare 1litre of 2x solution

10x Enzyme buffer :

  • 40 ml 100 mM citric acid

  • 60 ml 100 mM tri-sodium-citrate

  • Adjust to pH4.8). Store stock solution at 4 ° C.

  • Make 100ml

  • For working strength solution dilute 1:9 in water. We will do that at the course.

10% SDS in water

  • We will need 1.5ml

Salmon Sperm DNA

Sonicated or autoclaved DNA from salmon or herring sperm or E. coli (1µg/µl) Fragments should be 100-300bp long. Store in aliquots at –20 °C

  • We need about 200 ul

  • 50% dextran sulphate in water

  • We will need 1.5 ml

3M sodium acetate

  • We need about 20ul

Enzyme solution

  • 2% (w/v) cellulase from Aspergillus niger (Calbiochem, 21947, 4000units/g; final concentration: 80 units/ml)

  • 3% (v/v) pectinase from Aspergillus niger (solution in 40% glycerol, Sigma P4716, 450units/ml; final concentration 13.5units/ml).

  • Make up in 1x enzyme buffer.

  • Store in about 1.5ml aliquots at –20 °C      

  • We will need a15 tubes (about 20ml)

Fixations

If possible can you make fixation of banana roots from the side of pots of young plants.

Other plants that you have available could be treated the same, or if seeds are available germinate those. I will bring fixed roots, but it is better to have a back-up in your lab.

 The following is a suitable method for making fixations and preparation of root-tips (root tip) from various species with small to medium-size chromosomes. From our book adapted for banana and other tropical plants


ACCUMULATION AND FIXATION OF PLANT METAPHASE CHROMOSOMES

Reagents

  • Metaphase arresting agents:

  • 2 mM 8-hydroxyquinoline (for dicotyledonous plants, particularly those with small chromosomes such as Arabidopsis thaliana ). Can be stored in the dark at 4°C for several days to weeks.

  • Alcohol:acetic acid fixative: 3 parts 96% ethanol (or 100% methanol) to 1 part glacial acetic acid (prepare immediately before use; do not store for more than 30 min as the compounds degrade).

Material

  • For analysis of metaphase chromosomes, any tissue containing dividing cells can be used: Root tips from young seedlings, from newly grown roots at the edge of plant pots or hydroponic culture are all suitable.

  • Alternatively, flower buds, anthers, carpels or leaf or apical meristems can be used (Notes 2 and 3).

  • For germination of many seeds, put onto filter paper saturated with distilled water at 20-25°C in the dark and leave until roots are about 10-20 mm long (Note 1). Seeds of trees that grow long single roots are best germinated in a pot of vermiculite, while small seeds are best germinated under sterile conditions on agar minimal medium, e.g. Murashige and Skoog without sugar (Notes 3 and 4). Seed suppliers will give advice about germination of difficult species; moving between 4 °C and 25 °C every 3-14 days often assists germination.

  • Plants established in soil (e.g. trees such as oil palm) may produce actively growing roots within a few weeks when compost is applied on the surface around the stem (Note 4).

Method

The following steps are carried out in small glass or plastic containers (5-10 ml) or 1.5 ml microcentrifuge tubes. Use generous amounts of solutions: typically 1 ml per specimen. Material is transferred carefully by clean forceps or a pipette (Note 2).

  • To accumulate metaphases, treat excised root tips (5-20mm long) or other material with one of the metaphase arresting agents as follows (Note 3): 8-hydroxyquinoline for 1-2 h at room temperature, then 1-2 h at 4°C For banana select the 1-3mm thick roots with white to light yellow tips (do not use thin or thicker roots and do not take black roots) and they will need 2.5h at growing temp and 2.5h at 4°C. Other tropical plants with same thickness of roots will use same timing, seedlings and thinner roots probably 1.5 –2 hours only in each warm and cold.

  • Quickly blot material and transfer to fixative (Note 5).

  • Fix for at least 16 h at room temperature. If fixed material is to be kept (up to several months), leave for 2 h at room temperature and then transfer to new fixative (or 70% or 96% ethanol) and store at -20°C.

Notes

  • The response to the metaphase accumulation reagents is different from species to species and has to be established by trial and error. Some guidelines for choosing are given; Dolezel et al . (1992) discusses alternative reagents, including spindle poisons used a herbicides.

  • It is very important not to expose seedlings, roots and plants during germination and metaphase-arrest to chemicals and fumes, particularly fixatives (e.g. in a cold room also used for chemical storage) and to use clean labware with tight lids (disposable plastic is ideal), clean forceps, and distilled water.

  • Root tips from germinating seeds, and plants grown in controlled conditions, often show waves of cell division that may follow internal or environmental rhythms (e.g. light) or correlate with root length. At certain times, there may be no divisions at all, so it may be helpful to make several fixations.

  • Representative times are given. For best results fix material after different times of treatment, experiment with different reagents and check the mitotic index by making chromosome preparations. Treating material for too long in arresting agents, particularly colchicine, results in over-condensation of the metaphase chromosomes which might be desirable for counting chromosomes, but not for in situ hybridization where spatial resolution along chromosomes is wanted.

  • Fixative should note be contaminated with water, so careful blotting or an extra rinse in fixative is advised.

Things and amounts to take (up to 10 students)

Please prepare the following. Quantities are approximate,

  • 4 Eppendorfs Sigma Molecular biology grade water

  • 1x 1ml 100mM EDTA pH 8

  • 2x 1ml 20x SSC

  • 1x 1ml 10% SDS

  • 1x 20 ul Salmon Sperm DNA (1µg/µl)

  • 1x 20ul RNase stock

  • 1x 200 ul 50% dextran sulphate in water

  • 20 ul 3M sodium acetate

  • 1ml Tween-20 (crewcap eppendorf)

  • 3x empty 50 ml polypropylene centrifuge tubes (to put slides in)

  • 2x 1.5ml Antifade (screw cap eppendorfs)

  • 5x 1ml 1ug/ml DAPI

  • 2x 1ml 6ug/ml DAPI

  • 13ml 10x Enzyme buffer:

  • 10x 1.5ml Enzyme solution (screw cap eppendorfs)
    ( i )2% (w/v) cellulase from Aspergillus niger (Calbiochem, 21947, 4000units/g; final concentration: 80 units/ml)
    (ii) 3% (v/v) pectinase from Aspergillus niger (solution in 40% glycerol, Sigma P4716, 450units/ml; final concentration 13.5units/ml).

  • 10x 1.5ml 2 mM 8-hydroxyquinoline (screw cap eppendorfs)

  • 2 Boxes of acid cleaned slides

  • Box (100) of 18x18mm coverslips

  • Box of 24x40mm coverslips

  • Box of filter papers

  • Razor blades

  • 2x Good forceps

  • 8x syringe needles