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Mike Karampelias, Ricardo Tejos, Tomasz Nodzynski, Jiri Friml, Steffen Vanneste (VIB - Department of Plant Systems Biology, Ghent University - 9052 Ghent, Belgium;)Immunodetection is a valuable tool for cell biology research, that allows to determine the localization and expression levels of endogenous proteins. In plants, the application of this technique is hindered by waxy layers, cell wall and other obstacles. Current protocols work nicely for the immunolocalization of integral membrane proteins such as auxin transporting PINs. However, immunolocalization of peripheral membrane proteins, like clathrin, remained difficult. We tested how different parameters of the current protocol can be changed for better preservation of the signal of peripherally attached clathrin. We observed profound improvement by a simple modification at the fixation step. This improve the ability to immunodetect several markers. Because this protocol was only functioning for immunolocalization in young roots, we aimed to expand it for older, recalcitrant tissues. Via a combination of cell wall degrading enzymes we obtained high resolution immunolocalization in lateral root primordia. Together, these modifications allow to evaluate the subcellular localization of a wide range of markers in plant tissues circumventing the need of time-consuming crosses.