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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+structure+and+spatial+distribution+in+endosperm+of+high-amylose+rice+starch+granules+with+different+morphologies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradually+decreasing+starch+branching+enzyme+expression+is+responsible+for+the+formation+of+heterogeneous+starch+granules.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dek35+encodes+a+PPR+protein+that+affects+cis-splicing+of+mitochondrial+nad4+intron+1+and+seed+development+in+maize.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+structure+characteristics+to+form+ultrahigh+oil+content+in+rapeseed.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+VKS1+regulates+mitosis+and+cytokinesis+during+early+endosperm+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+comparison+and+applications+of+different+sections+in+investigating+the+microstructure+and+histochemistry+of+cereal+kernels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+whole+sections+of+mature+cereal+seeds+to+visualize+the+morphology+of+endosperm+cell+and+starch+and+the+distribution+of+storage+protein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+image+processing+method+for+investigating+the+morphology+of+cereal+endosperm+cells.">An image processing method for investigating the morphology of cereal endosperm cells.</a> Biotech &amp; Histochemistry. 95 (4), 249-261 (2020).</li><li>Borisjuk, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+architecture+shapes+embryo+metabolism+in+oilseed+rape.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+bodies+in+seeds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+endosperm+cells+and+starch+granules+in+common+wheat.">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 resin sections give a two-dimensional (2D) picture, leading to no way in analyzing the true size and quantity of cells. However, the cells are cut randomly at their any planes, the mean size of many cells (over 100) from at least three different sections of tissue region can reflect the 2D morphology parameters (length, width, and area) of cells, and the ratio of the chosen region area to mean cell area can reflect the quantity of cells. Therefore, it is very important for in situ viewing and analyzing the morphology of cells and storage materials in different regions of seed. The paraffin section is the most suitable for preparing the whole-seed-sized section, especially for large sized seeds7. However, the cells are full of storage materials with seed development, leading to that it is very difficult in obtaining the good whole-seed-sized section from late developing seeds and mature seeds. In addition, the paraffin section is too thick to exhibit the morphology clearly, and is only suitable for investigating the tissue structure of seed7.
The resin section is thin, and can exhibit the morphology of cells, starch granules, and protein bodies clearly7. However, the routine resin is not suitable for whole-seed-sized section. The technique presented here represents a fast, simple, and keen approach toward preparing transversal and longitudinal whole-seed-sized sections of mature seeds embedded in resin for viewing the morphology of cells, starch granules, and protein bodies in different regions of seed using light microscopy (Figure 2-5,&#160;Supplementary Figure 1). In addition, the technique can also prepare the section of developing, germinated, and cooked seeds to in situ investigate the morphology changes of cell, starch, and protein bodies in different regions of seed.
Another distinct advantage that this technique provides is the application of whole-seed-sized sections. In the new era of phenomics and metabolomics, it is important to quantitatively measure the morphology parameters of cells, starch granules, and protein bodies in different regions of seeds. The new technique, in conjunction with morphology analysis software, allows the researcher to quantitatively analyze the morphology parameters of cells, starch granules, and protein bodies in different regions of seed (Supplementary Table 1-4).
Though the present dry sectioning method can successfully prepare the whole-seed-sized resin section, it has some limitations and shortcomings. For the paraffin section, the paraffin can be removed easily from the section; but for the resin section, the resin cannot be removed from the section, leading to the plant sample embedded in resin. Therefore, compared with the paraffin section, the present whole-seed-sized resin section is not suitable to carry out the histochemistry and immunohistochemistry. In addition, the routine resin sectioning method can cut samples into 0.5-2 &#181;m smooth sections due to the sample block with small volume. But the present dry sectioning method is difficult to prepare the smooth sections with thickness less than 2 &#181;m, especially for mature seeds with large volume and high starch content.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.

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

Nanyang Technological University

Research

JoVE Journal

Biology

4.4K Views.  Yangzhou University. 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.

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

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

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 ...