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Immunolocalization analysis is a principal tool to study protein expression and subcellular distribution in plant cells or tissues. In this chapter, we present the method of the preparation of lightly fixed fresh rice leaf tissue for immunolocalization analysis and detection of the protein of interest using fluorescent probes by fluorescent microscopy. This method especially does not need the process of embedding plant materials that saves time and prevents alterations of cellular compounds and structure during sample preparation. Using this method, the C₄ rice project compared the expressions of the proteins of interest among C₄ model plants, wild-type rice, and transgenic or mutant plants and successfully selected the transgenic plants with the correct location of each protein to create a C₄ rice prototype.