Three-Dimensional Immunolocalization In Maize Meiocytes

Abstract

During meiotic prophase I, the physical configuration of chromosomes varies widely across developmental stages, fortunately these stage-specific configurations allow the proper identification of each of these classical meiotic stages, which are respectively known as leptotene (e.g. the ‘thin threads’), zygotene (e.g. the ‘coupled threads’), pachytene (e.g. the ‘thick threads’), diplotene (e.g. the ‘double threads’) and diakinesis (e.g. the ‘repulsion motion’). The visual analysis of several cytogenetic phenomena that occur during prophase I, such as recombination, pairing, and synapsis, requires highly reliable immunolocalization techniques. In contrast to traditional spreading and squashing techniques that suppress all subcellular and subnuclear information, the 3D conservation procedure described here allows the preservation of key meiotic proteins in their native subcellular location sites (cytoplasmic, nuclear, and subnuclear: euchromatin, heterochromatin, and nucleolar). Thus, this protocol describes how to efficiently fix meiotic anthers, dissect and release meiocytes from anthers, and perform simultaneous immunolocalization of three key proteins in maize male meiocytes.