Protocol

Preparation of Arabidopsis Tissue Sections of Fixed Material

This protocol was adapted from "How to Analyze a Mutant Phenotypically," Chapter 4, in Arabidopsis (eds. Weigel and Glazebrook). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.

INTRODUCTION

The best tissue sections are obtained from plastic-embedded material. These sections are more difficult and more expensive to prepare than those from Paraplast-embedded material, but the superior results make the additional effort worthwhile. The procedure starts with a fixative cocktail that includes a quickly penetrating fixative, such as paraformaldehyde, and one that is good for preserving fine structure, such as glutaraldehyde. Next, the samples are stained to facilitate manipulation of embedded tissue. After dehydration and staining, the tissue is infiltrated with resin. Finally, the embedded tissue is mounted and sectioned. The following protocol can be used on roots, leaves, shoot apices, and flowers, but the duration of infiltration must be adjusted for the various tissue types. Roots can be processed within 2 d, whereas leaves and flowers require up to 2 wk infiltration. Because Arabidopsis seedlings are small, whole seedlings (1-2 weeks old) can be fixed (including the root system) without dissection. Similarly, entire inflorescences, including those that have just opened, can be processed. Before embedding, the fixed material should be dissected to isolate organs of interest, such as individual flowers, to allow better orientation. Leaves from plants older than 2 wk should be cut into 1-cm² pieces and fixed individually.