Abstract
Confocal laser scanning microscopy in combination with fluorescent proteins is a powerful tool for the study of sexual reproduction and other developmental processes in plants. In order to understand the origin and localization of fluorescent signals in a complex tissue, staining of cell outlines is often mandatory. Cell wall staining with SCRI Renaissance 2200 (SR2200) has recently been described as a method of choice to study plant reproductive processes (Musielak et al., 2015). In this protocol, we present detailed instructions on the use of SR2200 to stain cell walls in different Arabidopsis tissues.
Keywords: Renaissance SR2200, Cell wall staining, Arabidopsis thaliana, Pollen tube, Confocal microscopy
Materials and Reagents
Equipment
Procedure
Representative data
Figure 3. Representative images of SR2200-stained Arabidopsis embryos. A. 8-cell embryo expressing pNMA>>NLS-tdtomato (red) (Babu et al., 2013). B. Late heart-stage embryo expressing Q0990>>erGFP (green) (Weijers et al., 2006). SR2200 signal in grey. C. Maximum projection of 2-cell embryo double-stained with SR2200 and DAPI. DAPI signal in nuclei (cyan) was separated from SR2200 signal in cell walls (grey) by spectral unmixing (Musielak et al., 2015). Scale bar = 20 µm.
Recipes
Acknowledgments
This protocol was adapted from the previously published study, Musielak et al. (2015). We would like to thank Christian Liebig and Aurora Panzera of the microscope facility at the MPI for Developmental Biology for technical assistance and support, Agnes Henschen and Laura Schenkel for their help in establishing the initial staining protocol. Research in our group is supported by the German Science Foundation (Deutsche Forschungsgemeinschaft-DFG: SFB1101/B01 to M.B.) and the Max Planck Society.
References
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