DETECTION OF IN SITU HYBRIDIZATION (ISH) SIGNALS WITH FLUOROCHROMES
- Remove cover slips by dipping the slides in a 2 X SSC solution and letting the cover slips fall from the slides.
- Wash slides by using the following steps (the temperature and time in the SSC treatment may be increased for reducing background).
| 2 X SSC at room temperature for 5 min, |
| 2 X SSC at 37°C for 10 min, |
| 2 X SSC at room temperature for 5 min, and |
| 1 X PBS at room temperature for 5 min |
- Drain (but never dry) slides on paper towels. Add 100-µL IgG fraction rabbit anti-biotin (Enzo Diagnostics #43861, 1:100 dilution) and cover with a '22 x 40' mm cover slip. Incubate at 37°C for 30 min.
- Remove cover slips by tilting the slides or dipping the slides in a beaker with 1 X PBS; wash slides three times in 1 X PBS (5 min each) at room temperature. The time and temperature of the washing can be changed to adjust the stringency.
- Remove cover slips by tilting or dipping the slides into a beaker containing 1 X PBS.
- Wash slides in 1 X PBS at room temperature for 5 min.
- Drain slides on paper towels, add a thin layer of antifade solution (10-mg/mL p-phenylenediamine in glycerol) containing 1-µg/mL propidium iodide, and cover with a cover slip.
- FITC and propidium iodide should be excited at 450-490 nm; the hybridization signals are yellow-green in color, whereas the chromosomes fluoresce red.
