Abstract
Immunolocalization studies to visualize the distribution of proteins on meiotic chromosomes have become an integral part of studies on meiosis in the model organism Arabidopsis thaliana. These techniques have been used to visualize a wide range of meiotic proteins involved in different aspects of meiosis, including sister chromatid cohesion, recombination, synapsis, and chromosome segregation. However, the analysis of meiotic spindle structure by immunofluorescence is of outstanding importance in plant reproductive biology and is very challenging. In the following report, we describe the complete and easy protocol for the localization of proteins to the male meiotic spindle and male meiotic chromosomes. The protocol is fast, improved, and robust without the use of any harsh enzymes.
Highlights
In plant biology, immunofluorescence is crucial for a variety of purposes, including protein localization and protein–protein interaction
A number of different techniques have been developed throughout the years to analyze meiotic chromosomes in Arabidopsis
One of the important microscopic studies that seem to be challenging is the analysis of spindle structure on meiotic chromosomes
Summary
Immunofluorescence is crucial for a variety of purposes, including protein localization and protein–protein interaction. We have set precise parameters for bud dissection and fixation and developed a protocol for reproducible visualization of spindle proteins during plant meiosis. A number of different techniques have been developed throughout the years to analyze meiotic chromosomes in Arabidopsis. A number of steps are followed, including cell permeabilization, flash freezing, squashing by physical force, enzyme digestion, and detergent treatment. One of the important microscopic studies that seem to be challenging is the analysis of spindle structure on meiotic chromosomes. The reported protocol allows robust immunolabeling of spindle fibers in Arabidopsis thaliana meiocytes, which enables higher resolution independent of the treatment of harsh enzymes. The protocol is the first-ever study to report the immunolocalization of meiotic chromosomal proteins without the use of enzymes
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