Procedure

1. Fix the tissue in FAA, embed in Paraplast, and section (as per in situ hybridization, see page 182)

2. Deparaffinize slides in Histoclear for 10 minutes. Repeat.

3. Hydrate through an ethanol series: 100%, 100%, 95%, 70%, 50%, 30%, distilled H20, distilled H20 for 2 minutes each.

4. Incubate the slides in 0.2 M HC1 for 20 minutes.

5. Incubate in distilled H.O for 5 minutes and then in 5x SSC for 5 minutes.

6. Incubate in 50 pg/ml proteinase K in 0.1 M Tris/0.05 M EDTA (pH 8.0) for 10 minutes.

The concentration of proteinase K or the length of treatment may need to be adjusted as different lots of proteinase K vary in specific activity.

7. Incubate in PBS for 10 minutes and then in BTX for 30 minutes.

8. Incubate in primary antibody diluted in BTX for 90 minutes at room temperature or overnight at 4°C.

To reduce background, primary antibody may be preabsorbed against acetone powder (Harlow and Lane 1999) from plant tissue, preferably from a protein-null mutant.

9. Wash the slides three times in BTX for 15 minutes each.

10. Incubate for 1 hour at room temperature with alkaline-phos-phatase-conjugated secondary antibody (Promega), diluted 1:1500 in BTX.

11. Wash the slides three times in BTX for 15 minutes each.

12. Develop with NBT/BCIP as described for DIG-labeled RNA probes (see page 201, Steps 8-10).

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