- Glass coverslips: No.0 24x40 or 24x50mm. (Note 4).
- Flat slide storage trays. The mountant remains fluid, so slides should be stored flat, in t he dark in a cold-room or refrigerator. Cardboard trays hold more slides than plastic and c an be stacked better, but decay in the humid conditions.
- McIlvaine's buffer (pH 7.0): 82 ml of 200 mM Na 2 HPO 4 and 18 ml 100 mM citric acid.
- 1xPBS (Appendix 1)
- Choose as required (Note 1)
(i)DAPI (4',6-diamidino-2-phenylindole, Sigma). Prepare DAPI stock solution of 100 µg/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 –20 °C (it is stable for years). Prepare a working solution of 2-4 m g/ml by dilution in McIlvaine's buffer, aliquot and store at –20 °C (Note 2 and 3).
(ii) PI (propidium iodide, Sigma). Prepare PI stock solution of 100 µg/ml in water. PI is a potential carcinogen. To avoid weighing out the powder, order small quantities and use the whole vial to make the stock solution. Aliquot 50 µl or 100 µl in 1.5 ml microcentrifuge tubes and store at –20 °C. Dilute with 1x PBS to 0.1-5 µg/ml (initially, use a low concentration such as 0.3 µg/ml) prior to use. PI does not keep in diluted form (Note 2).
- Antifade solution and mounting medium: these must be UV transparent and autofluorescence free.
(i)Commercial mounting media: e.g., Vectashield (Vector Laboratories), Slow Fade (Molecular Probes), Fluorguard (Sigma) or Citifluor AF1 (Agar)
(ii) To 60-90% (v/v) glycerol (specified for fluorescence) in 1x SSC (or 1x PBS or water) add one or more of the following :
- To 60-90% (v/v) glycerol (specified for fluorescence) in 1x SSC (or 1x PBS or water) add one or more of the following
- 0.2-0.5% PPD (2,5-diphenyl-1,3,4-oxadiazol; Sigma)
- 2 to 5% DABCO (1,4-diazabicyclo[2.2.2]octane; Sigma)
- 4% n-propyl gallate (NPG)
Store at 4°C; some solutions go brown with time but this does not effect their efficiency
5. Glass coverslips: No.0 24x40 or 24x50mm. (Note 4).
6. Flat slide storage trays. The mountant remains fluid, so slides should be stored flat, in the dark in a cold-room or refrigerator. Cardboard trays hold more slides than plastic and can be stacked better, but decay in the humid conditions.
Preparations following stringent washes (direct fluorophore labels, Protocol 9.1), detection of hybridization sites (indirect labels, Protocol 9.2) or in situ
primer extension (direct fluorophore labels, Protocol 8.5).
- Remove slides from the staining jar and drain excess buffer
- Put 100 m l DAPI or PI solution on the marked area of the preparation on each slide. Cover with a plastic coverslip and incubate at room temperature for 10 min, avoiding bright light (Note 2 and 3).
- Remove coverslips and wash slides briefly in detection buffer
- Add two drops (total about 80 µl) of antifade solution to each preparation and cover with a large, thin coverslip avoiding bubbles. Allow to settle for a few minutes and then gently, but firmly, squeeze excess antifade from the slide between several sheets of filter paper.
- Analyze preparations with an epifluorescence microscope (see Chapter 11)
- Take care that the counterstains complement the hybridization detection fluorophores: do not use DAPI with blue fluorophores or PI with red fluorophores. It may also be worth inspecting slides without counterstaining since some probes (e.g. genomic DNA) label all chromosomes enough to enable visualization. See Table 4.1 for properties and further choices of DNA stains.
- Avoid over-staining of chromosomes, which obscures weak hybridization. Concentration of stain not time of staining needs to be adjusted. Often, it is worth examining some slide unstained. PI can be washed out by removing the coverslip and soaking for 5 min to overnight in 4x SSC/Tween; washing also reduces DAPI staining.
- DAPI has two modes of binding to DNA: in the minor groove, which occurs at high stain concentrations; and, at lower DAPI concentrations, the alternative of intercalation between AT-nucleotide tracts in the DNA (Wilson et al. 1990). The latter binding gives the greater increase in fluorescence compared to the unbound molecule. Hence, increasing stain concentration has sometimes the unusual effect of reducing signal, and weak signal may mean too high stain concentration
- No.0 (0.1 mm) is thinner than the most widely used specification (No. 1, 0.13 – 0.16 mm) and is recommended for high resolution fluorescent microscopy