Mechanical fixation techniques for processing and orienting delicate samples, such as the root of Arabidopsis thaliana, for light or electron microscopy

Despite improvements in live imaging, fixation followed by embedding and sectioning for light or electron microscopy remains an indispensible approach in biology. During processing, small or delicate samples can be lost, damaged or poorly oriented. Here we present a protocol for overcoming these issues when, along with chemical fixation, the sample is fixed mechanically. The protocol features two alternatives for mechanical fixation: the sample is encased either in a rectangular block of agarose or between Formvar films suspended on a wire loop. We also provide methods for key steps all the way through to sectioning. We illustrate the method on the root of Arabidopsis thaliana, an object that is ～0.15 mm in diameter and difficult to process conventionally. With this protocol, well-oriented sections can be obtained with excellent ultrastructural preservation. The protocol takes about 1 week.     

Feulgen Staining of Intact Plant Tissues for Confocal Microscopy

A method was developed to prepare plant structures for confocal laser scanning microscopy by combining Feulgen staining with pararosaniline and embedding in LR White_TM. This procedure preserves intact, delicate structures for three-dimensional imaging without loss from sectioning or squashing, and the slides can be viewed several times without serious photobleaching.     

Feulgen Staining of Intact Plant Tissues for Confocal Microscopy

A method was developed to prepare plant structures for confocal laser scanning microscopy by combining Feulgen staining with pararosaniline and embedding in LR White_TM. This procedure preserves intact, delicate structures for three-dimensional imaging without loss from sectioning or squashing, and the slides can be viewed several times without serious photobleaching.     

New embedding medium for sectioning undecalcified bone.

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Fast x-ray fluorescence microtomography of hydrated biological samples

Metals and metalloids play a key role in plant and other biological systems as some of them are essential to living organisms and all can be toxic at high concentrations. It is therefore important to understand how they are accumulated, complexed and transported within plants. In situ imaging of metal distribution at physiological relevant concentrations in highly hydrated biological systems is technically challenging. In the case of roots, this is mainly due to the possibility of artifacts arising during sample preparation such as cross sectioning. Synchrotron x-ray fluorescence microtomography has been used to obtain virtual cross sections of elemental distributions. However, traditionally this technique requires long data acquisition times. This has prohibited its application to highly hydrated biological samples which suffer both radiation damage and dehydration during extended analysis. However, recent advances in fast detectors coupled with powerful data acquisition approaches and suitable sample preparation methods can circumvent this problem. We demonstrate the heightened potential of this technique by imaging the distribution of nickel and zinc in hydrated plant roots. Although 3D tomography was still impeded by radiation damage, we successfully collected 2D tomograms of hydrated plant roots exposed to environmentally relevant metal concentrations for short periods of time. To our knowledge, this is the first published example of the possibilities offered by a new generation of fast fluorescence detectors to investigate metal and metalloid distribution in radiation-sensitive, biological samples.     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin™ as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin™ for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Aerosol ot Solution—An Effective Softener of Herbarium Specimens for Anatomical Study

Dried plant parts are cut into convenient sizes and soaked in a solution containing 2.5-3.3% Aerosol OT in distilled water for 5 or more hours until well penetrated by the Aerosol. After brief washing in distilled water the material can be embedded, can be sectioned freehand or, if the nature of the material permits, with a sliding microtome, without embedding. Although it is a good wetting agent, Aerosol is chemically neutral; therefore, microchemical tests can be performed successfully on material treated with it. Refractory plant tissues embedded in paraffin can be successfully softened if one face of the block is trimmed to expose the tissue, then soaked in an Aerosol solution before sectioning.     

Zur Verwendung von 2-Hydroxyethyl-Methacrylat (GMA) als Einbettungsmedium bei histologischen Untersuchungen in der Phytopathologie

The use of 2-hydroxyethyl-methacrylate (GMA) as embedding medium for histological investigations in phytopathology A new plastic embedding technique is described for subsequent thin sectioning of plant tissues. In comparison to the paraffinmethod the GMA polymerization system is less time consuming. The excellent preservation of well-fixed tissue is fully asserted, as the embedding medium is not removed from the sections. In lightmicroscopic studies convincing results were obtained with different staining procedures; specific evidence for polysaccharides, pectine and nucleic acids was carried out with thin sections of 2-8 μm thickness, also by fluorescence microscopy. The GMA-embedding technique seems to be of value for various histological investigations in phytopathology.     

Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens

In performing in situ hybridizations, nonisotopic nucleic acid labeling coupled with colorimetric detection offers a safer, easier and more rapid alternative to using radioactively labeled nucleic acid probes and microscopic autoradiography. Whole mount in situ hybridization is also advantageous, because many samples can be processed identically and the reduced handling of specimens greatly reduces the risk of exposing tissues to RNase(s). The thickness of whole mount specimens, however, often prevents accurate determination of sites of expression within specific tissues. Although post-hybridization embedding and sectioning is a solution to this problem, the precipitate formed following the common colorimetric detection procedure is soluble in the organic solvents used for dehydration prior to embedding. We have developed a dehydration and embedding procedure that takes advantage of the compatibility of L.R. White^® resin containing 10% (v/v) polyethylene glycol 400, and heat polymerized. The addition of the plasticizer allows L.R. White^® embedded tissues to be sectioned at 10 μm providing excellent signal contrast.     

Orientation of Small Specimens for Cryosectioning

A simple technique is introduced to achieve symmetrically oriented frozen sections of small specimens such as young fish or frog larvae. Small samples are especially difficult to orient if they are already frozen to the chuck in a freezing microtome. Orientation of the sample in a mold filled with embedding medium prior to freezing permits sectioning as well as easy labeling and storage of the specimens. The use of a stereo microscope during orientation is optional.     

Spatially resolved analysis of small molecules by matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI)

? Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of tissues provides the means to analyse the spatial distributions of small molecules and proteins within tissues. This imaging technique is commonplace in medicinal and pharmaceutical research, but its application in plant science is very recent. Broader introduction requires specific adaptations for plant tissues. Sample preparation is of paramount importance in order to obtain high-quality spectra providing sufficient spatial resolution for compounds. Optimization is required for sectioning, choice of matrix and means of matrix deposition. ? Here, we present our current protocols for the detection of small molecules in cryodissected immature barley (Hordeum vulgare) grains and tobacco (Nicotiana tabacum) roots. ? Examples of MALDI-MSI measurements are provided, and the level of reproducibility across biological replicates is addressed. Furthermore, our approaches for the validation of distribution patterns and for the identification of molecules are described. ? Finally, we discuss how MALDI-MSI can contribute to applied plant research.     

Fluorescence in situ hybridization on vibratome sections of plant tissues

This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d.     

Adaptation of Cryo‐Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans

Cryo‐sectioning procedures, initially developed by Tokuyasu, have been successfully improved for tissues and cultured cells, enabling efficient protein localization on the ultrastructural level. Without a standard procedure applicable to any sample, currently existing protocols must be individually modified for each model organism or asymmetric sample. Here, we describe our method that enables reproducible cryo‐sectioning of Caenorhabditis elegans larvae/adults and embryos. We have established a chemical‐fixation procedure in which flat embedding considerably simplifies manipulation and lateral orientation of larvae or adults. To bypass the limitations of chemical fixation, we have improved the hybrid cryo‐immobilization–rehydration technique and reduced the overall time required to complete this procedure. Using our procedures, precise cryo‐sectioning orientation can be combined with good ultrastructural preservation and efficient immuno‐electron microscopy protein localization. Also, GFP fluorescence can be efficiently preserved, permitting a direct correlation of the fluorescent signal and its subcellular localization. Although developed for C. elegans samples, our method addresses the challenge of working with small asymmetric samples in general, and thus could be used to improve the efficiency of immuno‐electron localization in other model organisms.      

Immunocytochemical technique for protein localization in sections of plant tissues

There is a growing demand for methods that allow rapid and reliable in situ localization of proteins in plant cells. The immunocytochemistry protocol presented here can be used routinely to observe protein localization patterns in tissue sections of various plant species. This protocol is especially suitable for plant species with more-complex tissue architecture (such as maize, Zea mays), which makes it difficult to use an easier whole-mount procedure for protein localization. To facilitate the antibody-antigen reaction, it is necessary to include a wax-embedding and tissue-sectioning step. The protocol consists of the following procedures: chemical fixation of tissue, dehydration, wax embedding, sectioning, dewaxing, rehydration, blocking and antibody incubation. The detailed protocol, recommended controls and troubleshooting are presented here, along with examples of applications.     

Correction of distortion of histologic sections of arteries

Histologic sections of arteries can be used to generate three-dimensional (3D) geometric models and identify structural constituents. However, geometric distortions are introduced by fixation, embedding and sectioning; distortions which can, for example, lead to errors in stresses predicted by finite element models. We developed a method to measure and correct for distortions caused by acrylic processing and applied it to intact, healthy porcine coronary arteries. Micro-computed tomography was used to image arteries in the fresh and embedded states. Tissue blocks were sectioned, stained and imaged using a light microscope. Each section contained four registration marks used to determine strains introduced by sectioning and staining. Using these three image sets, 3D geometric models were generated and distortions were measured. Fixation, processing, and embedding resulted in shrinkage of 6.4+/-2.3% axially and 35.4+/-5.0% in mean cross-sectional area (n=5). Shrinkage in a cross section was well characterized by a uniform, equibiaxial strain. Sectioning and staining resulted in additional compressive strains in the sectioning direction of 0.067+/-0.011 and, in the direction perpendicular to sectioning, of 0.023+/-0.005 (n=5). These strains are assumed uniform and form the basis for correcting section geometry. Reconstructions using corrections for sectioning and shrinkage-related distortions had errors of 1.6+/-0.5% (n=5) and 4.0+/-1.7% (n=5), respectively.     

Modifications of a Method for Preparing Retinal Wholemounts for Sectioning

A method for embedding and sectioning retinal wholemounts is described. It employs a hydrophilic embedding agent, LR White, and coverslips inert to standard embedding resins. This simple technique, which has worked successfully on both enzymatically and autoradiographically labelled material, provides a means by which retinal wholemounts can be cut nearly parallel to the plane of the retinal layers. Previously prepared retinal whole-mounts can also be successfully embedded and sectioned more than a year later by the method described.     

Modifications of a method for preparing retinal wholemounts for sectioning

A method for embedding and sectioning retinal wholemounts is described. It employs a hydrophilic embedding agent, LR White, and coverslips inert to standard embedding resins. This simple technique, which has worked successfully on both enzymatically and autoradiographically labelled material, provides a means by which retinal wholemounts can be cut nearly parallel to the plane of the retinal layers. Previously prepared retinal wholemounts can also be successfully embedded and sectioned more than a year later by the method described.     

Standardization in immunohistochemistry: the role of antigen retrieval in molecular morphology

In performing in situ hybridizations, nonisotopic nucleic acid labeling coupled with colorimetric detection offers a safer, easier and more rapid alternative to using radioactively labeled nucleic acid probes and microscopic autoradiography. Whole mount in situ hybridization is also advantageous, because many samples can be processed identically and the reduced handling of specimens greatly reduces the risk of exposing tissues to RNase(s). The thickness of whole mount specimens, however, often prevents accurate determination of sites of expression within specific tissues. Although post-hybridization embedding and sectioning is a solution to this problem, the precipitate formed following the common colorimetric detection procedure is soluble in the organic solvents used for dehydration prior to embedding. We have developed a dehydration and embedding procedure that takes advantage of the compatibility of L.R. White^® resin containing 10% (v/v) polyethylene glycol 400, and heat polymerized. The addition of the plasticizer allows L.R. White^® embedded tissues to be sectioned at 10 μm providing excellent signal contrast.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.