- Acid-cleaned slides from protocol 4
- 18 X 18 mm (no.1) coverslips (Note 5)
- Dissecting needles and fine forceps
- 10x Enzyme buffer: 40 ml 100 mM citric acid + 60 ml 100 mM tri-sodium-citrate (10x stock, adjust to pH4.8). Store stock solution at 4 ° C. (For working strength solution dilute 1:10 in water for use)
- Enzyme solution: 2% (w/v) cellulase from Aspergillus niger (Calbiochem, 21947, 4000units/g; final concentration: 80 units/ml) or a mixture of 1.8% Calbiochem (72units/ml) and 0.2% 'Onozuka' RS cellulase (5000 units/g, final concentration 10units/ml) and 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 aliquots at –20 °C (Notes 1, 2, 3)
- 45% and 60% acetic acid: dilute glacial acetic acid with distilled water to the appropriate concentration
- Dry ice or liquid nitrogen
Fixed root tips, buds or other material from Protocol
Each of the following steps, unless stated, is carried out at room temperature in small glass or plastic containers (5-10 ml) or 1.5 ml microcentrifuge tubes. Material is carefully transferred by forceps or, if small roots or buds are used, with a pipette. Alternatively, fluids can be removed with a pipette and replaced with the next required solution
- Wash 2-10 root tips or buds twice for 10 min in 2-5 ml enzyme buffer to remove the fixative (until they sink).
- Transfer material into 1-2ml enzyme solution and digest at 37°C until the material is soft, usually 45 - 90 min (Notes 2 and 3)
- Wash material in enzyme buffer for at least 15 min
- Transfer enough material for one preparation (typically one root or small bud) into 45% acetic acid in an embryo dish or small Petri dish for 1-5 min
- Make chromosome preparations on an acid cleaned slide. Under the stereo microscope, in 1 drop (10-30 µl) of 45% acetic acid (or 60% acetic acid to increase dispersion of cytoplasm), dissect the meristematic tissue by removing as much of the other tissue as possible, e.g. remove the root cap and tease out the cells in the remaining terminal 1-3 mm (Note 6)
- Apply coverslip to the material without trapping air bubbles. Carefully disperse the material between glass slide and coverslip by tapping the coverslip gently with a needle or flat back of a pencil, and then squash the cells, usually using the thumb with a pressure that just turns the nail white (Note 7)
- Check the slide under a phase contrast microscope. If not sufficiently flat squash again
- Place the spread slide on to dry ice for 5-10 min (preferred method) or immerse into liquid nitrogen until frozen (about 30 sec), then flick off the coverslip with a razor blade (Note 8). Allow the slide to air-dry
- Screen slides to choose suitable preparations for in situ hybridization.
- Spread preparations can be stored desiccated for up to 3 months at 4 ° C or -20°C.
- Most protocols give enzymes concentrations as percentages; therefore we give units for the products we currently use. Onozuka cellulase is a very pure enzyme and has a constant activity per weight. In general, crude enzyme grades are used: purified enzymes (typically protoplast grades) have different properties and may not give as good preparations for in situ hybridization. Batches of the same enzyme product can differ in strength and composition, while distributors may change isolation protocols or suppliers. For example, pectinase from Sigma changes every two or three years, and they currently sell two (P0690 and P4716) with identical descriptions from different suppliers. With some companies the product number and description may not change although the properties of the product may change greatly .
- Enzyme mixtures can be reused several times: after use, centrifuge in a microcentrifuge, transfer the supernatant to a new tube, mark for reuse and freeze (not recommended for screening lines of similar material since cells may remain in the solution). Digestion time might need to be slightly increased after each round of use .
- Sometimes the ratio of cellulase to pectinase needs to be adjusted or different enzymes such as macerozymes, pectolyase or cytohelicase (for meiotic tissue see additional protocol) can be included. The enzyme digestion step needs to be adjusted to the material and species used, by changing the time of digestion; aim at a digestion time of 45-90 min otherwise change the strength of enzyme. Ideally, cell walls should be weakened, so that the cells can be separated easily, and chromosome spreads are clean of cytoplasm. In most cases, the meristematic cells will be digested faster than the non-dividing tissue. The material should remain intact to handle, otherwise the dividing cells are lost into the medium. If material has been fixed for several weeks, the material becomes harder and needs longer digestion .
- If dry ice or liquid nitrogen are not available, slides can be frozen on a metal plate in a -70 °C freezer .
- 18x18 mm glass coverslips of medium thickness are used. As well spread metaphases are often near the periphery of the coverslip, slightly larger coverslips are then used for hybridization and visualization (e.g. 24 x 24 mm) so that all cells can be probed and examined. The coverslips should be free of dirt, but should not be cleaned with alcohol or acid; otherwise material will stick to the coverslip and will be lost when the coverslip is removed .
- It is important to make a monolayer of cells and nuclei and to use the correct amount of fluid that is needed for squashing between the coverslip and glass slide (Step 6). Excess fluid will move all cells to the edge of the coverslip, too little fluid will encourage air bubbles. Metaphase cells tend to float away from cell clumps. Thick and too dense material will not spread. Also, be careful to remove any dirt or glass particles (e.g. from writing with a diamond pen), as they will prevent squashing
- Tapping with the needle on top of the coverslip disperses cells, but care needs to be taken not to shear the material by lateral movement of the coverslip. The final thumb pressure to squash the material should be moderately strong, but not abrupt
- If screening (Step 9) suggests that too much cytoplasm is present, alcohol:acetic acid fixative (3:1) at -20 °C can be dropped onto or next to the preparation from 10-50mm immediately after removal of the coverslip, before drying. This seems to disperse cytoplasm, especially in species with small chromosomes
- This spreading protocol relies on fixation of the material by precipitation in alcohol:acetic acid followed by chromosome spreading; the method was first developed in plants in the 1930s by CD Darlington and L LaCour ( Darlington , 1937), replacing complex sectioning methods and allowing very rapid advances in chromosome research. Acetic acid destroys many proteins, making the DNA within the chromosomes accessible to stains or probes. Acetic acid is also used to soften the material prior to spreading and allows chromosomes to stick to the glass slide. Removing of the coverslip follows quick freezing (Conger & Fairchild 1953). The protocol for digestion with enzymes is based on Schwarzacher et al . (1980) and produces excellent preparations even from old fixations.