Name: A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications (Video) | JoVE Journal
Uploaded: 2021-01-23T13:00:00.000+00:00
Duration: 10 min 10 s
Description: Learn a simple dry sectioning method for whole-seed-sized resin sections using LR White resin and maize kernels. This technique enables detailed visualization and quantitative analysis of cells, starch granules, and protein bodies.

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5 minutes

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

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 µm (Figure 2-5, Supplementary Figure 1-4). For examples, the mature seed of oilseed rape can be se...

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

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Learn a simple dry sectioning method for whole-seed-sized resin sections using LR White resin and maize kernels. This technique enables detailed visualization and quantitative analysis of cells, starch granules, and protein bodies.

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

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