1. Wash slides in 2x SSC, 50% formatnide: Remove coverslips by soaking slides in wash buffer at 50-55°C. Then wash twice for 1 hour at 50-55°C.

As with hybridization, the optimal temperature for washing will depend on the probe.

2. Wash slides for 5 minutes at room temperature in TBS.

3. Incubate in the blocking agent provided in the Roche DIG kit (0.5% in TBS; heat to 60°C to dissolve) for 1 hour.

From this point on, use Coplin jars instead of staining dishes to minimise buffer volume.

4. Wash in 1% BSA, 0.3% (v/v) Triton X-100 in TBS for 30 minutes.

5. Dilute anti-DIG antibody 1:3000 in 1% BSA, 0.3% (v/v) Triton X-100 in TBS. Incubate the slides for 90 minutes.

6. Wash in 1% BSA, 0.3% (v/v) Triton X-100 in TBS, three times for 20 minutes each.

7. Equilibrate the slides for 5 minutes in detection buffer.

8. Add 150 pi of NBT (100 mg/ml) and 150 pi of BCIP (50 mg/ml) to 100 ml of detection buffer and fill jars. Incubate in the dark. Check under dissecting microscope after 12 hours. Incubate for a total of up to 36 hours or longer.

9. Stop the enzyme reaction in TE.

10. To mount slides: Dehydrate sides through an ethanol series (90 seconds each), followed by Histoclear, twice for 90 seconds. Mount slides with Permount.

Double Labeling

It is sometimes desirable to detect the expression of two genes on the same tissue section. This can be achieved by labeling one probe with DIG-conjugated ribonucleotides and a second with fluorescein isothio-cyanate (FITC)-conjugated ribonucleotides. Both probes are included in the same hybridization solution, but detection is in two steps. The following steps describe the procedure.

1. Label one probe with DIG-rUTP and the other with FITC-12-rUTP (Roche Molecular Biochemicals). Include both probes in the hybridization solution.

2. Detect the DIG-labeled probe first following the regular protocol (see above). After signal development, wash the slides -n TE and mount them in distilled H20 with a coverslip. Photograph to record the single expression pattern. Do not allow the slides to dry out.

There are many variations on the final order of label detection that can be used. Detection of DIG first is given as an example.

3. Inactivate alkaline phosphatase by incubating slides in lx SSC ?n a rack for 2 hours in at 65°C.

4. Take the slides through a second antibody reaction using the anti-FITC antibody conjugated to alkaline phosphatase. After washing, the alkaline phosphatase substrate Fast Red, which comes in tablet form (Roche Molecular Biochemicals), is used. The substrate must be very fresh; prepare and change at least two to three times a day. When an adequate signal is detectable using a dissecting microscope (typically after 12-36 hours), stop the reaction in distilled H20. Ethanol may dissolve the red precipitate, so mount slides in water, glycerol, or another aqueous mounting medium.

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