Stringency Washes and RNase Treatment

These steps help to remove the nonspecific binding of the probe.

All buffers should be preheated to the washing temperature.

1. First wash: transfer the sections from the 24-well plate to washing vials containing solution A and wash twice for 15 min at room temperature. The first wash is discarded as radioactive waste.

3. Third wash: twice for 1 h at 55°C in solution C.

5. Finally rinse thoroughly with PBS and then with solution E at room temperature.

The washed hybridized sections can be either mounted directly on microscope slides as described below, or used for immunohistochemistry before mounting (see Subheading 4.5.).

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