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.

Analytical staining of cellulosic materials: A review

“…The term immunological implies that one takes advantage of a highly specific interaction between an antibody and an antigen protein, usually with one of them attached to a fluorescent chromophore or "label". There are many examples of such staining for the analysis of lignocellulosic materials or fibers (Xiao et al 1999;Hutterer et al 2017;Paes et al 2017;Verhertbruggen et al 2017).…”

“…For example, Lux et al (2005) recommended the addition of lactic acid saturated with chloral hydrate as a clearing agent for living plant tissues. Another common procedure is embedding, in which the fragile living tissue becomes reinforced by the curing of an added material (Verhertbruggen et al 2017). Such procedures can allow the specimen to be microtomed into thin sections for examination of the three-dimensional aspects.…”

Characterization of Selected Plants Leaves with Particular Emphasizes on Epidermis

“…View under the compound light microscope at an appropriate magnification (usually 100x or 400x). In an unstained epidermis it helps to close the iris diaphragm a little to increase contrast and see the cell walls clearly [12]. The limitations of this method includated not all leaves are easy to peel and frequently mesophyll cells come off with the epidermal layer, giving more than one cell layer and making identification of the epidermal cells difficult and confusing…”

Report on the Current Inventory of the Toolbox for Plant Cell Wall Analysis: Proteinaceous and Small Molecular Probes

“…mAbs are also usually a researcher's first choice for use in in situ analysis (Hervé et al, 2011 ; Avci et al, 2012 ; Verhertbruggen et al, 2017 ). mAbs can be used either with whole mounts, for instance, for surface labeling of Arabidopsis roots (Larson et al, 2014 ) or pollen tubes (Chebli et al, 2012 ) on fresh hand- or vibratome-sectioned material, or on embedded and (ultra-, cryo-) microtome sectioned material.…”

“…There are considerable methodological differences between the various methods for sectioning and their applications to address particular scientific questions. These have been extensively compared by Verhertbruggen et al ( 2017 ). The large size of mAbs is sometimes an obstacle for accurate analysis, even in very thinly sectioned material.…”