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A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy.

ABSTRACT: Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of these three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.

SUBMITTER: Verhertbruggen Y

PROVIDER: S-EPMC5581911 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The micro-morphology of leaf epidermises is valuable for the study of leaf development and function, as well as the classification of plant species. There have been few studies comparing different preparation and imaging methods for visualizing the leaf epidermis. Here, four specimen preparation methods were used to investigate the leaf epidermis morphology of Arabidopsis, radish, cucumber, wheat, rice, and maize, under an inverted basic light microscope (LM), a laser scanning confocal microscope (LSCM), or a scanning electron microscope (SEM). Optical microscope specimens were obtained using either the direct isolation method or the chloral hydrate-based clearing method. SEM images were obtained using a standard stage for conventional dehydrated samples or a Coolstage for fresh tissue. Different parts of epidermis peels were well focused under the LM. Investigation of samples cleared by chloral hydrate is convenient and autofluorescence of cell walls can be detected in rice. The resolution of images of conventional SEM leaf samples was generally higher than the Coolstage images at the same magnification, whereas local collapse and shrinkage were observed in leaves with high water content when using the conventional method. However, stomatal apparatuses of Arabidopsis, cucumber, radish, and maize deformed and showed poor appearance when using the Coolstage. Moreover, we usually used glutaraldehyde as an SEM fixative when using t-butanol for freeze-drying, though methanol is considered a better fixative in recent studies. In addition, fresh samples were not stable on the Coolstage. Thus, we compared four different t-butanol freeze-drying methods and two Coolstage methods. The dimension and morphology of tissues were compared using the six different methods. The results indicate that methanol fixative obviously reduced shrinkage of SEM samples compared with glutaraldehyde and formaldehyde alcohol acetic acid (FAA) fixatives. The use of methanol and a graded series of steps improved the preservation of samples. Preparing samples with optimal cutting temperature compound and observing at -30°C helped to increase the stability of Coolstage samples. In summary, our results provide an overview of the shortcomings and merits of four different methods, and might provide some information about choosing an optimal method for visualizing epidermal morphology.

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A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy.

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