Histochemistry of Single Molecules

Malgorzata Ciska, Susana Moreno Díaz de la Espina

Carlo Pellicciari, Marco Biggiogera

At present, two complementary approaches are used for in situ protein visualization in plant nuclei. Imaging of transformed fluorescent proteins is the election tool for the analysis of protein movement and interaction. However, this methodology presents several drawbacks for the identification/localization of endogenous nuclear factors, such as over-expression or mislocalization of transformed proteins. In contrast, immunocytochemistry with specific antibodies represents a powerful tool for the localization of endogenous nuclear proteins at their "native" nuclear sub-compartments. In plant cells, the cell wall hampers antibody accessibility during immunocytochemical analysis thereby reducing the effectivity of the technique, particularly in the case of lowly expressed proteins. To overcome this problem in nuclear protein immunodetection, we developed a method based on the in vitro incubation of isolated nuclei with specific antibodies followed by imaging by confocal fluorescence or electron microscopy. Here we describe the application of this methodology to the localization of Nuclear Matrix Constituent Proteins (NMCP), the plant analogs of lamins, of the monocot Allium cepa, using antibodies raised against highly conserved regions of the proteins.