Radioactive In Situ Hybridization

This protocol is adapted from one used in the Meyerowitz, Weigel, and Yanofsky laboratories. The procedure consists of seven parts: Fixation, Embedding, Sectioning, Probe Synthesis, Hybridization, Washing, and Autoradiography.

PART 1: Fixation Materials

Tissue samples High-grade ethanol (100%) Ethanol series (60%, 70%, 85%, 95%) Glacial acetic acid Formaldehyde (37% v/v)

Xylene or xylene substitute such as Histoclear (Fisher Scientific,

Taab Laboratories) or Hemo-De (Fisher Scientific) Paraplast Plus chips Eosin Y (Sigma)

CAUTION: ethanol, formaldehyde, glacial acetic acid, xylene (see Appendix 3) Procedure

IMPORTANT As with the other methods in this section, avoid RNase contamination at all times. All water must be RNase-free.

1. Prepare FAA fixative as follows. For 100 ml:

ethanol 50 ml glacial acetic acid 5 ml

37% formaldehyde 10 ml distilled H20 35 ml

2. Place several pieces of tissue (such as small leaves or flower clusters) in 10-15 ml of fixative in 20-ml scintillation vials.

3. Place vials in a desiccator. Pull a mild vacuum very slowly, until bubbles begin to emerge. After 15 minutes, release the vacuum very slowly. Repeat. Incubate the vials for ~4 hours at room temperature.

Tissue will usually sink to the bottom after the second vacuum. If the tissue does not sink, repeat.

4. Remove fixative and add 50% ethanol to the sample vials. Incubate for 30 minutes at room temperature. Repeat.

5. Move samples through an ethanol series (60%, 70%, 85%, 95%) for 30 minutes at each concentration.

6. Leave the samples overnight in 95% ethanol with 0.1% eosm Y.

The stain makes the t sue easier to see during sectioning.

7. The following day, replace the 95% ethanol with 100% ethanol and incubate for 1 hour.

The tissue destains slowly in 100% ethanol, so do not incubate for an extended period (e.g., overnight).

8. Remove as much of the 100% ethanol as possible and replace it with fresh 100% ethanol. Incubate for 30 minutes at room temperature. Repeat this step if virtually all of the ethanol solution was not removed in previous steps.

9. Replace the ethanol with 25% xylene:75% ethanol. Incubate for a further 30 minutes at room temperature.

IMPORTANT Perform xylene steps in a chemical fume hood.

10. Repeat Step 9 for the following xyleneiethanol solutions: 50% xylene:50% ethanol; 75% xylene:25% ethanol.

11. Replace the 75%:25% solution with 100% xylene and incubate for 1 hour at room temperature. Repeat twice. Half-fill the scintillation vial with xylene the final time.

12. Add -20 Paraplast chips to each vial. Incubate overnight at room temperature. If time is short, proceed to the next step, omitting overnight incubation. For the next step, fill a beaker with Paraplast chips and place at 60°C; it will take at least 5 hours to melt. About 100 ml of molten Paraplast is needed for each vial of samples.

13. The following morning, the Paraplast in xylene will be in solution only partially. Place the vials in a 42°C incubator for -30 minutes to dissolve the chips completely.

14. Add -20 more chips, and incubate at 42°C until dissolved (—30 minutes). Swirl occasionally. Repeat until the vial is full (four to five times, -100 chips total).

15. Remove the xylene/Paraplast solution and replace with molten Paraplast. Swirl to mix. Incubate for at least 4 hours at 57-62°C. Repeat at least four times.

FART 2: Embedding

This protocol is greatly facilitated by the use of an embedding station

(e.g, Leica EG1160 or Microm AP280), but these are quite expensive.

The following protocol assumes that an embedding station is not available.

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