Detection

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Detection of ISH Signals with Fluorochromes

1.     Peel the rubber cement using a forceps.  Dip the slides in a staining jar containing 2X SSC.  Gently shake the staining jar until the coverslips fall from the slides.

2.     Wash the slides with the following steps:

        2X SSC, room temperature                    5 min

        2X SSC, 42°C                                         10 min

        2X SSC, room temperature                    5 min

        1X PBS, room temperature                    5 min

        Notes: You may increase the temperature and time of the 2X SSC wash if you want to reduce background.  The above washing series works fine for most of the probes in our hands.

3.     Drain (never dry) the slides on paper towels and add 100 µl of FITC (or rhodamine) conjugated anti-biotin (or anti-digoxigenin) antibody (1:50 to 1:200 dilution depending on the product from different companies) cover with 22 ´ 40 mm coverslips, incubate at 37°C for 30 min.

4.     Remove the coverslips by tilting the slides or dipping the slides in a beaker with 1X PBS.  Wash the slides three times in 1X PBS (5 min each) at room temperature.  Notes: You can change the time and temperature for the washing to adjust the stringency.

5.     Drain the slides on paper towel.  Add a thin layer of antifade solution (from Vector) containing 1 µg/ml propidium iodide or DAPI (4’,6-diamidino-2-phenylindole), cover with a coverslip.

6.     Check your in situ hybridization results using a fluorescence microscope.  The FITC-detected probes will be in a yellow-green color.  The rhodamine-detected probes will be a red color.