Fluorescence in situ hybridization on vibratome sections of plant tissues

Abstract

This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d. INTRODUCTIONMany of the most interesting aspects of plant cell biology and development occur in cells deep within tissues of the plant. Examples are male meiosis occurring within the anthers, and embryogenesis and endosperm development occurring within the developing seed. These cells are difficult or impossible to image, even within confocal microscopy, which is rarely able to image deeper than 100 mm. Thus to be accessible to imaging, dissection or sectioning must be used. Vibratome tissue sectioning is a simple way to produce relatively thick sections (20-50 mm), which can be imaged to reveal the structure of the underlying tissues. It has the advantage of preserving 3D structures well, so that subcellular organization can be reliably imaged. Furthermore, reliable identification of cell types often requires an accurate assessment of the tissue context, which is lost when cytological squash preparations are made.This method does not require embedding of tissue in wax or resin, and can be applied to fixed or unfixed tissues. The only requirements are that the plant tissue has to be sufficiently rigid for sectioning, and the plant parts have to be large enough to handle in the vibratome. These requirements are met by the roots and florets and other organs of many plants. We have used it for both roots and florets of wheat (Triticum aestivum L.) and related species 1-3 , for rye (Secale cereale L.) florets 4 , for rice (Oryza sativa L.) roots and florets 5 and maize (Zea mays L.) roots 6 among the cereals, and for roots of pea (Pisum sativum L.), bean (Vicia faba L.) 7 and soya (Glycine max L.) 8 . The only plant species with which we have been unsuccessful in vibratome sectioning is Arabidopsis thaliana, because of its small size. By carrying out the sectioning in 100%

Effective chromosome pairing requires chromatin remodeling at the onset of meiosis

“…(29). The anthers were sectioned in water with a vibratome, and the 50-to 100-m sections were placed onto a ␥-aminopropyl triethoxy silane (APTES)-coated slide that had been glutaraldehyde (2.5%)-activated (29). Slides were dried overnight at room temperature.…”

“…However, because both Cdk2 and Ph1 affect replication we hypothesize that the sensing mechanism occurs during premeiotic replication. (29). The anthers were sectioned in water with a vibratome, and the 50-to 100-m sections were placed onto a ␥-aminopropyl triethoxy silane (APTES)-coated slide that had been glutaraldehyde (2.5%)-activated (29).…”

In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)

“…The application of the BAC-FISH method to pericarp tissue necessitated the development of a new protocol adapted from that of Prieto et al (2007). We had to optimize several parameters -tissue preservation, section thickness, the method for handling the sections and permeabilization steps.…”

“…Indeed, dehydration (step 5) was performed before denaturation (step 4) which was realized with the following modifications: after dehydration, denaturation of samples and probes was achieved simultaneously at 78°C for 8 min (according to Prieto et al, 2007). This longer denaturation step appeared to be much more effective than the 70% formamide treatment for 2 min at 70°C used for isolated nuclei.…”

“…Fixed tissues were then washed for 10 min in 50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), 150 mM NaCl (pH 7) (TBS) and 150 lm sections were obtained using a vibrating blade microtome Microm HM 650V (Microm MicroTech France, http://mm-france.fr/). Very slow and large-amplitude sectioning was performed as recommended by Prieto et al (2007). The pericarp sections were treated according to two slightly different protocols:…”