Cleared Tissue for Observation of Vascular Strands

This protocol was contributed by Sioux Christensen (Salk Institute).

Vascular strands constitute an internal tissue that is important for plant growth. The strands are easily imaged by whole-mount analysis. It is well established that the hormone auxin has an important role in the formation of vascular strands (Mattson et al. 1999; Sieburth 1999). For a discussion of mutations that specifically disturb vascular development, see Carland et al. (1999) and Deyholos et al. (2000).

Materials

Tissue sample

Ethanol/acetic acid mixture (6:1) Ethanol (70% and 100%)

Chloral hydrate/glycerol/water mixture 8:1:2 (g:ml:ml) CAUTION: acetic acid, chloral hydrate, ethanol (see Appendix 3)

Procedure

1. Fix the tissue sample in ethanol/acetic acid mixture at room temperature, 1-4 hours for embryos and 8-24 hours for seedlings and older or thicker tissues.

2. Remove ethanol/acetic acid mixture, replace with 100% ethanol, and incubate for 30 minutes at room temperature. Repeat.

3. Remove 100% ethanol, replace with 70% ethanol, and incubate for 30 minutes.

4. Remove ethanol and replace with chloral hydrate/glycerol/water mixture.

5. Clear the sample. Allow at least 1 hour for younger tissues. Allow older tissues, including rosette leaves, stems, and flowers, to clear overnight at room temperature.

6. Store cleared material at room temperature. It will become more fragile during storage but should remain stable for a few weeks.

7. Mount the sample in chloral hydrate mixture and view under dark-field illumination. Watch for vascular defects such as increased vascular bundles and discontinuity in vascular strands.

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