Electron microscopic immunogold localization of recombination-related proteins in spreads of synaptonemal complexes from tomato microsporocytes.
Many of the structures involved in meiotic synapsis and recombination such as synaptonemal complexes (SCs) and recombination nodules (RNs) can be resolved only by electron microscopy. Therefore, electron microscopic (EM) immunolocalization using gold-conjugated antibodies is the best way to verify whether certain proteins are components of SCs or RNs. Here, we describe (1) preparing tomato primary microsporocyte protoplasts in leptotene, zygotene, and pachytene stages; (2) hypotonically bursting the protoplasts on glow-discharged glass and plastic-coated slides to make spreads of SCs; (3) immunolabeling proteins in SCs and RNs with colloidal gold; (4) staining SC spreads for EM; and (5) transferring SC spreads on plastic films to grids for EM.
Stack
Stephen M
SM
Department of Biology, Colorado State University, Fort Collins, CO, USA.
Anderson
Lorinda K
LK
D007150:Immunohistochemistry; D018551:Solanum lycopersicum / Q000166:cytology / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D008540:Meiosis / Q000235:genetics / Q000502:physiology; D008854:Microscopy, Electron / Q000295:instrumentation / Q000379:methods*; D010940:Plant Proteins / Q000032:analysis / Q000378:metabolism*; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D011995:Recombination, Genetic / Q000502:physiology*; D013573:Synaptonemal Complex / Q000378:metabolism*; D016707:Tissue Fixation / Q000379:methods
D010940:Plant Proteins