Abstract
ImmunoFISH is a method combining immunolabelling (IL) with fluorescent in situ hybridisation (FISH) to simultaneously detect the nuclear distribution of proteins and specific DNA sequences within chromosomes. This approach is particularly important when analysing meiotic cell division where morphogenesis of individual proteins follows stage-specific changes and is accompanied by a noticeable chromatin dynamism. The method presented here is simple and provides reliable results of high quality signal, low background staining and can be completed within 2 days following preparation. Conventional widefield epifluorescent or laser scanning microscopy can be used for high resolution and three-dimensional analysis. Fixation and preparation techniques were optimised to best preserve nuclear morphology and protein epitopes without the need for any antigen retrieval. Preparation of plant material involved short cross-linking fixation of meiotic tissues with paraformaldehyde (PFA) followed by enzyme digestion and slide-mounting. In order to avoid rapid sample degradation typical of shortly fixed plant materials, and to be able to perform IL later, slides were snap-frozen and stored at -80°C. Ultra-freezing produced a remarkable degree of structural preservation for up to 12 months, whereby sample quality was similar to that of fresh material. Harsh chemicals and sample dehydration were avoided throughout the procedure and permeability was ensured by a 0.1–0.3% detergent treatment. The ImmunoFISH method was developed specifically for studying meiosis in Triticeae, but should also be applicable to other grass and plant species.
Highlights
Chromosome–chromosome interactions leading to homologous pairing at meiosis are accompanied by programmed changes in chromatin organisation and structure
Chromatin dynamics could be mapped in high resolution within the meiotic nuclei of bread wheat
Our technique produces a single layer of well-preserved, cytoplasm-free nuclei embedded in antifade, an ideal situation for high resolution imaging with confocal laser scanning microscopy (CLSM)
Summary
Chromosome–chromosome interactions leading to homologous pairing at meiosis are accompanied by programmed changes in chromatin organisation and structure. Distinct stages of chromatin remodelling and movement with association of landmark chromosomal regions imprint the period of early meiosis and result in a distinctive nuclear polarisation (Colas et al, 2008; Heslop-Harrison and Schwarzacher, 2011; Sepsi et al, 2017). To understand regulation and interdependence of these complex events, and to evaluate possible effects on plant fertility, the morphogenesis of specific proteins needs to be visualised in the context of chromatin organisation and movement. Methods are available to track meiotic proteins in Arabidopsis (Chelysheva et al, 2010; Higgins et al, 2014) but optimal tissue preservation and achievement of high-quality immunosignal in grasses with their large chromosomes (30–100 times the size of Arabidopsis; Choulet et al, 2010; Heslop-Harrison and Schwarzacher, 2011) requires modifications of fixation, preparation and labelling techniques
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