Funding was provided by the National Natural Science Foundation of China (32071927), the Talent Project of Yangzhou University and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

1. Preparation of resin-embedded seed (Figure 1)
<ol>
	<li>Fix six maize mature kernels in 10 mL of 2.5% phosphate-buffered glutaraldehyde (0.1 M, pH7.2) at 4 &#176;C for 48 h. The researchers can choose other fixative mixtures, fixative concentrations, and fixation conditions according to their research objectives and tissue types.</li>
	<li>Take out the kernels and slice them longitudinally or transversally to 2-3 mm thickness using a sharp double-sided blade, and fix them in 10 mL of 2.5% phosphate-buffered glutaraldehyde (0.1 M, pH 7.2) again for 48 h.</li>
	<li>Wash the samples three times with 10 mL of 0.1 M phosphate buffer (pH 7.2) for 30 min every time.</li>
	<li>Dehydrate the samples in increasing grades of ethanol aqueous solution (10 mL) from 30% to 50%, 70%, 90% once, and 100% three times for 30 min every time.</li>
	<li>Infiltrate the samples in 10 mL increasing grades of LR White resin solution diluted with ethanol from 25% to 50%, 75% once, and 100% twice at 4 &#176;C for 12 h every time.</li>
	<li>Prepare the pedestals for samples before embedding. Add 0.25 mL of 100% LR White resin into a 2-mL centrifuge tube, and polymerize it at 60 &#176;C for 48 h.</li>
	<li>Successively add the pure LR White resin (0.5 mL) and the infiltrated sample into the centrifuge tube with a pedestal. Straighten the samples with the anatomical needle, and polymerize them at 60 &#176;C in an oven for 48 h.</li>
</ol>
2. Dry sectioning for preparing whole-seed-sized section (Figure 1)
<ol>
	<li>Take out the embedded kernels from the centrifuge tube and cut out the excess resin around the sample using a sharp blade.</li>
	<li>Clamp the resin block in the sample holder of ultramicrotome (EM UC7), and trim off the superfluous resin on the surface of the sample and around the sample with a blade.</li>
	<li>Polish the surface of the sample finely with a glass knife until a complete section can be formed.</li>
	<li>Put a small copper hook about 2 mm above the blade edge before cutting and cut the sample into a 2 &#181;m section. The role of the hook is to avoid the curling upward of the section.</li>
	<li>Put the hook under the section to support it when the section becomes long.</li>
	<li>Add 100 &#181;L of water on an unpretreated slide, and carefully transfer the complete and unbroken section to the water with the tweezers.</li>
	<li>In order to smooth the wrinkled section, heat and dry the sample on the flattening table at 50 &#176;C overnight.
	<ol>
		<li>If the section crumbles or tears, extend the time for each resin infiltration of the sample from 12 h to 24 h or 48 h.</li>
		<li>If the section has some lines paralleled to the knife, clamp the sample block tightly. If the section has some lines vertical to the knife, please use a new knife.</li>
	</ol>
	</li>
</ol>
3. Staining and observation of the section
NOTE: In order to observe the tissue structure and morphology of cells, starch granules, and protein bodies, stain the sections with specific stains according to the purpose of the research. Here, we use the fluorescent brightener 28, iodine solution, and Coomassie brilliant blue R250 to stain the cell walls, starch granules, and protein bodies, respectively.
<ol>
	<li>For observing the morphology of cells, stain the section with 40 mL of 0.1% (w/v) fluorescent brightener 28 aqueous solution in a 70 mL compact glass staining jar at 45 &#176;C for 10 min, and then rinse it with running water for 5 min. Observe and photograph the section under a fluorescence microscope equipped with a CCD camera.</li>
	<li>For observing the morphology of starch granules, stain the section with 40 &#181;L of iodine solution (0.07% (w/v) I2 and 0.14% (w/v) KI in 25% (v/v) glycerol) for 1 min, and cover the sample containing iodine solution with a coverslip. View and photograph the sample under a light microscope equipped with a CCD camera.</li>
	<li>For observing the morphology of protein bodies, immerse the section with 40 mL of 10% (v/v) acetic acid in a 70 mL compact glass staining jar for 10 min at 45 &#176;C, and then stain it in 40 mL of 1% (w/v) Coomassie brilliant blue R250 in 25% (v/v) isopropanol and 10% (v/v) acetic acid for 15 min at 45 &#176;C. Wash the stained sections with running water for 5 min, and dry it. Observe and photograph the section under a light microscope equipped with a CCD camera.</li>
</ol>
4. Quantitative analysis of morphology parameters
<ol>
	<li>Process and quantitatively analyze the photographed images for area, long/short axis, and roundness of cells, starch granules, and protein bodies in different regions of seed using morphology analysis software (Image-Pro Plus 6.0 software) following the procedures of Zhao et al.9 exactly.</li>
</ol>

<ol><li>Cai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneous+structure+and+spatial+distribution+in+endosperm+of+high-amylose+rice+starch+granules+with+different+morphologies%5BTitle%5D)+AND+%22Journal+of+Agricultural+and+Food+Chemistry%22%5BJournal%5D)">Heterogeneous structure and spatial distribution in endosperm of high-amylose rice starch granules with different morphologies.</a> Journal of Agricultural and Food Chemistry. 62 (41), 10143-10152 (2014).</li>
<li>He, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+defective+effect+of+starch+branching+enzyme+IIb+from+weak+to+strong+induces+the+formation+of+biphasic+starch+granules+in+amylose-extender+maize+endosperm%5BTitle%5D)+AND+%22Plant+Molecular+Biology%22%5BJournal%5D)">The defective effect of starch branching enzyme IIb from weak to strong induces the formation of biphasic starch granules in amylose-extender maize endosperm.</a> Plant Molecular Biology. 103 (3), 355-371 (2020).</li>
<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gradually+decreasing+starch+branching+enzyme+expression+is+responsible+for+the+formation+of+heterogeneous+starch+granules%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Gradually decreasing starch branching enzyme expression is responsible for the formation of heterogeneous starch granules.</a> Plant Physiol. 176 (1), 582-595 (2018).</li>
<li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dek35+encodes+a+PPR+protein+that+affects+cis-splicing+of+mitochondrial+nad4+intron+1+and+seed+development+in+maize%5BTitle%5D)+AND+%22Molecular+Plant%22%5BJournal%5D)">Dek35 encodes a PPR protein that affects cis-splicing of mitochondrial nad4 intron 1 and seed development in maize.</a> Molecular Plant. 10 (3), 427-441 (2017).</li>
<li>Hu, Z. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Seed+structure+characteristics+to+form+ultrahigh+oil+content+in+rapeseed%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Seed structure characteristics to form ultrahigh oil content in rapeseed.</a> PLoS One. 8 (4), 62099(2013).</li>
<li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maize+VKS1+regulates+mitosis+and+cytokinesis+during+early+endosperm+development%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Maize VKS1 regulates mitosis and cytokinesis during early endosperm development.</a> Plant Cell. 31 (6), 1238-1256 (2019).</li>
<li>Xu, A., Wei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+comparison+and+applications+of+different+sections+in+investigating+the+microstructure+and+histochemistry+of+cereal+kernels%5BTitle%5D)+AND+%22Plant+Methods%22%5BJournal%5D)">Comprehensive comparison and applications of different sections in investigating the microstructure and histochemistry of cereal kernels.</a> Plant Methods. 16, 8(2020).</li>
<li>Zhao, L., Pan, T., Cai, C., Wang, J., Wei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+whole+sections+of+mature+cereal+seeds+to+visualize+the+morphology+of+endosperm+cell+and+starch+and+the+distribution+of+storage+protein%5BTitle%5D)+AND+%22Journal+of+Cereal+Science%22%5BJournal%5D)">Application of whole sections of mature cereal seeds to visualize the morphology of endosperm cell and starch and the distribution of storage protein.</a> Journal of Cereal Science. 71, 19-27 (2016).</li>
<li>Zhao, L., Cai, C., Wei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+image+processing+method+for+investigating+the+morphology+of+cereal+endosperm+cells%5BTitle%5D)+AND+%22Biotech+%26+Histochemistry%22%5BJournal%5D)">An image processing method for investigating the morphology of cereal endosperm cells.</a> Biotech & Histochemistry. 95 (4), 249-261 (2020).</li>
<li>Borisjuk, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Seed+architecture+shapes+embryo+metabolism+in+oilseed+rape%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">Seed architecture shapes embryo metabolism in oilseed rape.</a> The Plant Cell. 25 (5), 1625-1640 (2013).</li>
<li>Lott, J. N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+bodies+in+seeds%5BTitle%5D)+AND+%22Nordic+Journal+of+Botany%22%5BJournal%5D)">Protein bodies in seeds.</a> Nordic Journal of Botany. 1, 421-432 (1981).</li>
<li>Jing, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+endosperm+cells+and+starch+granules+in+common+wheat%5BTitle%5D)+AND+%22Cereal+Research+Communications%22%5BJournal%5D)">Development of endosperm cells and starch granules in common wheat.</a> Cereal Research Communications. 42 (3), 514-524 (2014).</li>
</ol>

The authors have nothing to disclose.

The seeds are the most important renewable resource for food, fodder, and industrial raw material, and are rich in storage materials such as starch and protein. The morphology and quantity of cells and the content and configuration of storage materials affect the weight and quality of seeds7,12. Though the stereology and image analysis technology can measure the size and quantity of cells in a tissue region, they are lacking in many laboratories. The paraffin and...

Plant seeds contain storage materials such as starch and protein and provide energy and nutrition for people. The shape, size, and quantity of cell and storage materials determine the weight and quality of seed. The cells and storage materials in different regions of seed have significantly different morphologies, especially for some high-amylose cereal crops with inhibition of starch branching enzyme IIb1,2,3. Therefore, it is very important to investigate the morphologies of cells and storage materials in different regions of seed.
Paraffin sectioning is a good method to prepare the whole-seed-sized section and can exhibit the tissue structure of seed and the accumulation of storage material in different regions of seed4,5,6. However, the paraffin sections usually have 6-8 &#181;m thickness with low resolution; thus, it is very difficult to clearly observe and quantitatively analyze the morphology of cell and storage materials. The resin sections usually have 1-2 &#181;m thickness and high resolution and are very suitable to observe and analyze the morphology of cell and storage materials7. However, the routine resin sectioning method has difficulty in preparing the whole-seed-sized section, especially for seeds with a large volume and high starch content; thus, there is no way to observe and analyze the morphology of cells and storage materials in different regions of the seed. LR White resin is an acrylic resin and exhibits low viscosity and strong permeability, leading to its good applications in preparing the resin section of seeds, especially for cereal mature kernels with large volume and high starch content. In addition, the sample embedded in LR White resin can be stained easily with many chemical dyes to clearly exhibit the morphology of cells and storage materials under light or fluorescent microscope7. In our previous paper, we have reported a dry sectioning method for preparing the whole-seed-sized sections of mature cereal kernels embedded in LR White resin. The method can also prepare the whole-seed-sized section of developing, germinated and cooked cereal kernel8. The obtained whole-seed-sized section has many applications in micromorphology observation and analysis, especially for clearly viewing and quantitatively analyzing the morphology differences of cell and storage materials in different regions of seed8,9.
This technique is appropriate for researchers who want to observe the microstructure of tissue and the shape and size of cells, starch granules, and protein bodies in different regions of seed using light microscope. The images of whole-seed-sized sections stained specifically for exhibiting cells, starch granules, and protein bodies can be analyzed by morphology analysis software to quantitatively measure the morphology parameters of cells, starch granules, and protein bodies in different regions of seed. In order to demonstrate the technical applicability and whole-seed-sized section applications, we have investigated the mature seeds of maize and oilseed rape and the developing, germinated, and cooked kernels of rice in this study. The protocol contains four processes. Here, we use mature maize kernel, which is the most difficult in preparing the whole-seed-sized sections due to the large volume and high starch content, as a sample to exhibit the processes step by step.

<table><tbody><tr><td>Acetic acid</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A501931</td><td></td></tr><tr><td>Compact glass staining jar (5-Place)</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>E678013</td><td></td></tr><tr><td>Coomassie brilliant blue R-250</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A100472</td><td></td></tr><tr><td>Coverslip</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>F518211</td><td></td></tr><tr><td>Double-sided blade</td><td>Gillette Shanghai Co., Ltd.</td><td>74-S</td><td></td></tr><tr><td>Ethanol absolute</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A500737</td><td></td></tr><tr><td>Flattening table</td><td>Leica</td><td>HI1220</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Olympus</td><td>BX60</td><td></td></tr><tr><td>Fluorescent brightener 28</td><td>Sigma-Aldrich</td><td>910090</td><td></td></tr><tr><td>Glass strips</td><td>Leica</td><td>840031</td><td></td></tr><tr><td>Glutaraldehyde 50% solution in water</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A600875</td><td></td></tr><tr><td>Glycerol</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A600232</td><td></td></tr><tr><td>Iodine</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A500538</td><td></td></tr><tr><td>Isopropanol</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A507048</td><td></td></tr><tr><td>Light microscope</td><td>Olympus</td><td>BX53</td><td></td></tr><tr><td>LR White resin</td><td>Agar Scientific</td><td>AGR1281A</td><td></td></tr><tr><td>Oven</td><td>Shanghai Jing Hong Laboratory Instrument Co.,Ltd.</td><td>9023A</td><td></td></tr><tr><td>Potassium iodide</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A100512</td><td></td></tr><tr><td>Slide</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>F518101</td><td></td></tr><tr><td>Tweezers</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>F519022</td><td></td></tr><tr><td>Sodium phosphate dibasic dodecahydrate</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A607793</td><td></td></tr><tr><td>Sodium phosphate monobasic dihydrate</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A502805</td><td></td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>EM UC7</td><td></td></tr></tbody></table>

a simple dry sectioning method for obtaining whole-seed-sized resin section and its applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

Simple dry sectioning method for obtaining a whole-seed-sized section 
We establish a simple dry sectioning method for preparing a whole-seed-sized section of seed embedded in LR-white resin (Figure 1). The method can prepare transversal and longitudinal whole-seed-sized sections with thickness of 2 &#181;m (Figure 2-5, Supplementary Figure 1-4). For examples, the mature seed of oilseed rape ca...

Watch this Scientific Journal Video about A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications at JoVE.com

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are ...

a-simple-dry-sectioning-method-for-obtaining-whole-seed-sized-resin

Research

JoVE Journal

Biology

4.5K Views.  Yangzhou University. De morfologiewetenschap en de kwaliteit van cellen, zetmeelkorrels en de eiwitlichamen in de kernel bepalen het gewicht en de kwaliteit van zaad. In deze studie presenteren we als een eenvoudige populatie van een hele kernel ingebed in LR Witte hars en stellen we een eenvoudige droge zuigmethode van hele pitten. We kiezen LR White hars als embedded medium om twee belangrijke redenen.Ten eerste is het lage viscositeit en sterke doorlaatbaarheid, wat leidt tot zijn goede toepassingen bij...

Video: A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

Environment

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Neuroscience

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

Engineering

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Biochemistry

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial companies have devised complex section transfer systems using fluid water. To simplify this technology, we created a simple method using homemade equipment that combines cutting and floating within a simple thermostatic chamber; therefore, the sections automatically enter the water bath on the water surface. The hippocampus from adult mouse brains, adult mouse kidneys, embryonic mouse brains, and adult zebrafish eyes were cut using both conventional paraffin sectioning and the presented method for comparison. Statistical analysis shows that our improved method saved time and produced higher quality sections. In addition, paraffin sectioning of a whole specimen in a short time is easy for junior operators.

Here, we present a protocol to improve paraffin sectioning. This method combines cutting and floating using a simple thermostatic chamber to avoid the transfer process required by the conventional method. As a result, the efficiency and the number of intact paraffin sections were greatly improved.

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

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