- 2x SSC: dilute from 20x SSC stock. RNase solution: DNase-free ribonuclease A (e.g. Sigma R4642, solution in 10 mM Tris-HCl, pH 8 and 50% glycerol, 70 units/mg protein). Make up stock of 10 mg/ml in 10 mM Tris-HCl, pH 8 (Appendix 1). Store at –20 °C in aliquots. Dilute to 100µg/ml prior to use (Note 1 and 2)
- 10 mM HCl
- Pepsin (from porcine stomach mucosa, e.g. Sigma P6887, 3200 to 4500 units/mg protein). Make up stock of 500 µg/ml in 10 mM HCl and store in aliquots at –20 °C. Dilute with 10mM HCl to 1-10 µg/ml prior to use (Note 3)
- Primers: For labeling inserts in M13 and related plasmids with the same multiple cloning site (e.g. pUC18, pUC19 or pBluescript) use the universal M13 forward and reverse sequencing primers: 5'GTA AAA CGA CGG CCA GT 3' and 5'GGA AAC AGC TAT GAC CAT G 3' or variants (Note 1). The stock primer solutions should be diluted to 10 µM
- Paraformaldehyde fixative: In the fume hood, add 4 g of paraformaldehyde (EM grade) to 80 ml water, heat to 60 °C for about 10 min, clear the solution with a few drops of concentrated (10 M) NaOH, let cool down and adjust the final volume to 100 ml with water. Adjust pH to 8 with 1N H 2 SO 4 (the solution has no buffering capacity so changes pH rapidly). The paraformaldehyde solution is best prepared freshly (during the Rnase and pepsin incubations), but can be stored for a few days at 4 °C
- Ethanol: 96%, 90% and 70%
- Good quality selected slides from preparation protocols (spreads, cytospin, whole mounts and sections, Chapters 5 and 6). Check using phase-contrast microscopy for amount of remaining cytoplasm (to decide time and concentration of any pepsin treatment needed), write numbers on slides, and mark the area of interest on each slide by scratching underneath with a diamond pen. Pencil writing on frosted-end slides, but not marker pens, will survive the washes and temperatures. It can be very difficult to identify the sample side and area as slides are placed into and removed from washes, but the scratches can be seen and felt easily. The time since fixation for material used for the preparation, and the storage conditions of the slide preparations, affect the length of pretreatments required (less for young fixations; note 2), as well as denaturation times and temperatures. We recommend keeping slides overnight at 37 °C after preparation, and then storing at -20°C for longer (up to several months). Some authors report better results from slides stored 2 weeks at room temperature.
For incubations on the slide, an ample volume, typically 200 µl, is applied to the sample area and covered with a large plastic coverslip to avoid drying out. For the washing steps, slides are put into staining jars and covered totally with solution (typically 80-100ml for up to 8 slides). Agitation by gently moving the staining jars by hand (once every minute) or continuously on a shaking platform is recommended. To change solutions, pour off carefully and replace slowly with next solution, or transfer slides to another staining jar with the next solution. Do not let slides dry out between changes of solutions. Carry out steps at room temperature if not otherwise stated
- The slides may be re-fixed and cleaned, particularly if stored for more than a week, by putting into alcohol:acetic acid fixative for 10 min, washing twice in 96% ethanol for 10 min each, and air drying
- Add 200µl RNase to the marked area of each slide, cover with a large plastic coverslip and incubate for 1 h at 37 ° C in a humid chamber (Note 2). Start preparing paraformaldehyde fixative
- Remove the coverslips carefully and wash slides in 2x SSC twice for 5 min
- Incubate slides in 10 mM HCl for 5 min. Take each slide, shake off excess fluid and quickly add 200 m l of pepsin, cover with a plastic coverslip and incubate for 10-15 min at 37 ° C in a humid chamber (Note 3).
- Remove the coverslips and stop the reaction by placing the slides in distilled water for 1 min. Wash slides in 2x SSC twice for 5 min .
- In the fume hood, place the slides in paraformaldehyde fixative for 10 min
- Wash in 2x SSC twice for 5 min
- Dehydrate slides through an ethanol series (70%, 90% and 96% ethanol, 2 min each). Air-dry in a rack. Do not dehydrate sections or whole mount preparations; go to Step 10
- Check dry slides by phase contrast microscopy. Cytoplasm should be removed by the pepsin treatment, but chromosomes should not be lost or damaged. (Note 4)
- Proceed to denaturation and hybridization (Protocol 8.4) or in situ primer extension (Protocol 8.5). If necessary, slides can be kept overnight at 4°C
- The RNase must be DNase-free. If RNase is not purchased DNase-free, inactivate DNase in the RNase by placing the stock solution in boiling water for 15 min
- RNase treatment is not essential for all materials and probes: RNA may be degraded already, and is not required for non-transcribed targets without RNA homology. Step 3 (acid wash) also degrades extraneous RNA
- Pepsin should be used to help remove excess cytoplasm covering chromosome preparations. Adjust concentration to suit the preparations; if there is little cytoplasm, no pepsin treatment is needed. Most spread preparations are treated with 1 µg/ml for 10 min, but up to 10 µg/ml for 1 h can be used; however, it is often better to improve the preparation technique (see Protocol 5.3 and 5.5, e.g. the use of additional 3:1 alcohol:acetic acid treatments or 60% acetic acid). For sections or whole mounts, proteinase K is more effective to remove cytoplasm (see Alternative Protocol below), and can also be used with spreads
- If loss of cells and chromosomes has occurred, it suggests that the slides were not acid-washed (Protocol 5.1) before spreading of chromosomes, and most likely further loss will occur. It is better to stop the procedure and start with new preparations
Notes to Table 8.1
- The formamide, SSC concentration and temperature determine the stringency of the hybridization (Chapter 7). We normally use final concentrations of 50% formamide and 2x SSC, which allows sequences of 75-80% homology to form duplexes. For genomic in situ hybridization, we often find that 40 µl hybridization solution is too little to fit all components with 50% formamide/2x SSC, so use 40% formamide and 1x SSC which also has a stringency of 75-80%
- For differentiation, each probe needs to carry a different label, eg. biotin, digoxigenin or a fluorophore. Examples shown use two cloned probes or two genomic probes. More or only one probe may be used, and cloned and genomic probes can also be combined. Probe concentration is approximate
- Excess amounts of unlabeled blocking DNA – genomic DNA or reannealed Cot=1 DNA – are used to out-compete hybridization of probe to undesired target sequences (see Section 3.1, genomic DNA and Section 7.3). Appropriate multiples of probe concentration to try are: CISS for human chromosomes, 2x probe; blocking clones, 10x probe; genomic DNA probe for detection of genomes in plant hybrids, 50x. If genomic probes are combined with cloned probes it is often necessary to increase the cloned probe concentration or reduce blocking DNA so hybridization of the cloned probe is not affected.