DETECTION OF IN SITU HYBRIDIZATION (ISH) SIGNALS WITH FLUOROCHROMES

1. Remove cover slips by dipping the slides in a 2 X SSC solution and letting the cover slips fall from the slides.
2. 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

1. 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.
2. 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.
3. Remove cover slips by tilting or dipping the slides into a beaker containing 1 X PBS.
4. Wash slides in 1 X PBS at room temperature for 5 min.
5. 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.
6. FITC and propidium iodide should be excited at 450-490 nm; the hybridization signals are yellow-green in color, whereas the chromosomes fluoresce red.