The authors acknowledge funding from the USDA-NIFA award 2016-67013-24816.

1. Preparation of experimental materials
<ol>
	<li>Prepare oatmeal agar (OMA) by blending oatmeal until it is a fine powder. Add 25 g of oatmeal powder and 15 g of agar to 500 mL of ddH2O, and autoclave on media cycle (alternatively bring to a boil for 20 min). Pour the media into sterile 60 mm Petri dishes. 
	NOTE: Other media types that induce sporulation, such as V8 agar, are acceptable for this protocol.</li>
	<li>Plate M. oryzae filter stocks directly onto the OMA plates using sterile forceps, and allow them to cover the entire plate (9-12 days). Place the plates in a growth incubator at 25 &#176;C with 12:12 h day:night cycles to help induce sporulation. 
	NOTE: Some mutants grow more slowly and require additional care (e.g., complete media first, then a transfer to the OMA), and could take an additional week to produce enough conidia.</li>
	<li>Directly plant barley (Hordeum vulgare Lacey) seeds in a moist growth medium (e.g., soilless potting media) with 10-15 seeds per 6 inch pot. Place the pots in trays with 1-2 in of water.</li>
	<li>Set the barley growth chamber conditions as 22 &#176;C for 12 h (daylight) and 19&#160;&#176;C for 12 h (dark) at 60% relative humidity. Continue to water from the bottom so as not to disturb the seeds.</li>
	<li>Grow the barley until the second leaf stage, approximately for 14 days. Using sterile scissors, cut the barley plant just above the soil line. Using forceps and a razor/scalpel, carefully cut the leaf sheath of the first leaf that is open longitudinally, and using the forceps, remove it from the base of the second leaf. 
	NOTE: Clean the tools (forceps, scalpel, etc.) before use with 75%-80% ethanol. The sheath is the thin epidermis layer that provides the attachment from the first to the second leaf (second to third leaf, etc.; see Figure 1).</li>
	<li>Place the first leaf flat in a sterile 60 mm Petri dish, containing a wet paper towel to maintain humidity inside the plate. Using the scalpel, cut the majority of the first leaf away from the sheath, leaving only 0.5 in of the leaf tissue for mounting.</li>
	<li>Tape the leaf tissue to the bottom of the Petri plate. 
	NOTE: The sheath curls, but this is acceptable as a curled sheath holds the conidial droplet more easily.</li>
	<li>Collect 9-12-day-old M. oryzae plates, and add 0.5-2 mL of sterile water to the plates. Using a sterile inoculation loop, gently scrape the mycelia to release the attached conidia. Carefully pipette the conidial suspension into a microcentrifuge tube containing a small piece of cheesecloth to filter out any large pieces of mycelium from the conidial suspension. 
	NOTE: Spores can be collected as early as 7 days, if growth and sporulation are sufficient to reach the desired spore concentration. Collection can be delayed no more than 14 days if working with a slow-growing genetic mutant</li>
	<li>The desired spore concentration is 5 x 104 spores per mL, but a range (1 x 104-1 x 105) is acceptable. Too high of a concentration makes imaging individual infection sites challenging; dilute the spore concentration with sterile water if necessary.</li>
	<li>Carefully pipette the conidial suspension inside of the rolled leaf sheath. Start with 25 &#181;L (the droplet size can be increased depending on the size of the sheath, up to 50 &#181;L). 
	NOTE: It is recommended to perform three to five replicates of each mutant strain or barley line. It is common for damage to occur during the staining process, therefore additional sheath replicates are recommended.</li>
	<li>Fill four or five 500 mL beakers with ddH2O, and heat until steaming (using a microwave or hotplate). Use caution when moving the hot water beakers. Hold the lid of the Petri dish over one of the steaming beakers to trap humidity inside the plate.</li>
	<li>Stack the infected leaf sheath plates and surround them with the remaining hot beakers. This creates a humid, moist environment, required for the spores to germinate. 
	NOTE: Use caution when steaming the lids to ensure no hot water or steam touches the sheaths.</li>
	<li>Protect the leaf sheaths from light, cover with a solid colored (black preferred) rubber or plastic box, and let sit for 48 h or the desired time-point for imaging. 
	&#8203;NOTE: A cardboard box is not suitable because it does not lock in the moisture/humidity and absorbs the steam from the hot water beakers. A locking, plastic tub works well for containing the humidity, and it can be covered with black fabric or a larger dark container to block the light.</li>
</ol>
2. Staining process
<ol>
	<li>Prepare the stain as follows: prepare a fresh dilution of 45% acetic acid and add 0.1% v/v trypan blue. Aliquot 1 mL of the dye solution into microcentrifuge tubes. Set a heat block or water bath to 40 &#176;C.</li>
	<li>Carefully, using a razor or scalpel, cut the leaf sheath away from the tape. Using forceps, place the sheath into the microcentrifuge tube and ensure that it is completely submerged in the dye solution. Allow 2 h for the dye to penetrate the leaf. Heat the samples at 40 &#176;C&#160;during the staining process time in a heat block or water bath to increase the penetration of the dye. 
	NOTE: The sheaths try to float in the micro-centrifuge tube; to prevent unstained pockets of leaf tissue, fill the tubes, submerge the sheaths, and close the tube. Do not put multiple sheaths in the same micro-centrifuge tube.</li>
	<li>Rinse the leaf sheaths carefully in 60% glycerol to remove the extra dye. Three rinses (each in fresh glycerol) are generally sufficient. Keep the sheath in glycerol until ready to mount on slides.</li>
</ol>
3. Mounting and imaging process
<ol>
	<li>Place the sheath on a clean glass slide and add a few drops of 60% glycerol. Using a dissecting microscope and two pairs of forceps, carefully unroll the sheath, leaving the inoculated center facing up. Hold the sheath open with the forceps, and place the coverslip on top to prevent the sheath from curling and blocking the infection site. 
	NOTE: The leaf sheath is very fragile, and these steps need to be done with care to prevent damage to the sheath.</li>
	<li>Seal the coverslip using nail polish for long-term storage, or tape for short-term storage. Observe the slides under a compound light microscope.</li>
	<li>Take basic images using a microscope and a cell phone. Here, images were taken with a cell phone adapter mount and a smartphone. For Android devices, adjust the camera application to the following settings: flash off, disable Top Shot,disable automatic adjustment of brightness and shadows, and set the photo resolution to full. 
	NOTE: Using the camera application on the phone decreases the battery life faster than usual, so having external power is recommended.</li>
	<li>Once the cell phone is mounted on the microscope, take an image of a scale micrometer with the objective that will be used to acquire the data. The data in this study was acquired at a 40x 0.65 NA air objective, and the phone adapter was mounted on a 10x ocular. Adjust the zoom of the phone to 2.5x, and keep it consistent to maintain a constant pixel size.</li>
	<li>The center of the sheath houses the largest concentration of spores and infecting appressoria; therefore, aim for 9-12 images of each sheath to obtain significant numbers for statistical analysis. The number of spores and appressoria vary based on the concentration of spores applied.</li>
</ol>
4. Image assessment and counting using ImageJ (FIJI)
<ol>
	<li>Transfer the images to a computer running ImageJ (FIJI). To open the images, drag and drop the files onto the ImageJ bar.</li>
	<li>Set the scale of the images by loading the stage micrometer image and drawing a straight line between two markings for the scale. Open Set Scale, type in the Known Distance for the line measured, and type in the unit for the scale. On the micrometer, in this example, the smallest line was 10 &#181;m. Check the Set Global box and hit OK. All subsequent images loaded will have the same scale.</li>
	<li>To count appressoria, spores, or other objects, select the Point tool. Next, open the ROI Manager. Click the T key on the keyboard to add points to the list. These regions of interest can be saved if needed and reloaded onto the same image.</li>
	<li>Depending on the experimental goals, make additional measurements, such as spore length, appressoria size, and germ tube length.</li>
</ol>

<ol>
	<li>Roy, K. K., 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=First+report+of+barley+blast+caused+by+Magnaporthe+oryzae+pathotype+Triticum+(MoT)+in+Bangladesh.">First report of barley blast caused by Magnaporthe oryzae pathotype Triticum (MoT) in Bangladesh.</a> Journal of General Plant Pathology. 87 (3), 184-191 (2021).</li><li>Dean, R., 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+Top+10+fungal+pathogens+in+molecular+plant+pathology.">The Top 10 fungal pathogens in molecular plant pathology.</a> Molecular Plant Pathology. 13 (4), 414-430 (2012).</li><li>Dean, R. A., 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+genome+sequence+of+the+rice+blast+fungus+Magnaporthe+grisea.">The genome sequence of the rice blast fungus Magnaporthe grisea.</a> Nature. 434 (7036), 980-986 (2005).</li><li>Wilson, R. A., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Under+pressure:+investigating+the+biology+of+plant+infection+by+Magnaporthe+oryzae.">Under pressure: investigating the biology of plant infection by Magnaporthe oryzae.</a> Nature Reviews. Microbiology. 7 (3), 185-195 (2009).</li><li>Giraldo, M. 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=Two+distinct+secretion+systems+facilitate+tissue+invasion+by+the+rice+blast+fungus+Magnaporthe+oryzae.">Two distinct secretion systems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzae.</a> Nature Communications. 4, 1996 (2013).</li><li>Islam, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+wheat+blast+in+Bangladesh+was+caused+by+a+SouthAmerican+lineage+of+Magnaporthe+oryzae.">Emergence of wheat blast in Bangladesh was caused by a SouthAmerican lineage of Magnaporthe oryzae.</a> BMC Biology. 14 (1), 84 (2016).</li><li>Langridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+and+Academic+Importance+of+Barley.">Economic and Academic Importance of Barley.</a> The Barley Genome. Compendium of Plant Genomes. , 1-10 (2018).</li><li>Heath, M. C., Valent, B., Howard, R. J., Chumley, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+two+strains+of+Magnaporthe+grisea+with+rice,+goosegrass,+and+weeping+lovegrass.">Interactions of two strains of Magnaporthe grisea with rice, goosegrass, and weeping lovegrass.</a> Canadian Journal of Botany. 68 (8), 1627-1637 (1990).</li><li>Gowda, M., 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=Genome+analysis+of+rice-blast+fungus+Magnaporthe+oryzae+field+isolates+from+southern+India.">Genome analysis of rice-blast fungus Magnaporthe oryzae field isolates from southern India.</a> Genomics Data. 5, 284-291 (2015).</li><li>Luginbuehl, L. H., El-Sharnouby, S., Wang, N., Hibberd, J. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+Genetic+Control+of+Reactive+Oxygen+Species+in+Magnaporthe+oryzae+(the+Rice+Blast+Fungus)+through+Development,+Screening,+and+Characterization+of+a+Random+Insert+Mutant+Library.">Identifying Genetic Control of Reactive Oxygen Species in Magnaporthe oryzae (the Rice Blast Fungus) through Development, Screening, and Characterization of a Random Insert Mutant Library.</a> University of Delaware. , (2022).</li><li>Zhang, M., 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=al.The+plant+infection+test:+Spray+and+wound-mediated+inoculation+with+the+plant+pathogen+Magnaporthe+grisea.">al.The plant infection test: Spray and wound-mediated inoculation with the plant pathogen Magnaporthe grisea.</a> Journal of Visualized Experiments. (138), e57675 (2018).</li><li>Koga, H., Dohi, K., Nakayachi, O., Mori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+inoculation+method+of+Magnaporthe+grisea+for+cytological+observation+of+the+infection+process+using+intact+leaf+sheaths+of+rice+plants.">A novel inoculation method of Magnaporthe grisea for cytological observation of the infection process using intact leaf sheaths of rice plants.</a> Physiological and Molecular Plant Pathology. 64 (2), 67-72 (2004).</li><li>Hamer, J. E., Howard, R. J., Chumley, F. G., Valent, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+for+surface+attachment+in+spores+of+a+plant+pathogenic+fungus.">A mechanism for surface attachment in spores of a plant pathogenic fungus.</a> Science. 239 (4837), 288-290 (1988).</li><li>Khang, C. 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The authors have nothing to disclose.

There are many commonly used assays available to test M. oryzae strains that provide a macroscopic-level visual of a compatible or incompatible infection response, such as spray or droplet inoculations, and the use of rating systems to quantifylesion sizes13,14. Another common assay for M. oryzae is to test the ability of the pathogen to form its specialized penetration structure, the apppressorium15. Described here is an...

Magnaporthe oryzae, the rice blast fungus, infects an assortment of grain crops, including barley, wheat, and rice1. This pathogen causes devastating diseases and poses a worldwide threat to these valuable crops, causing complete crop loss if not controlled. Many labs around the world focus on rice blast disease because of its global threat and its attributes as an excellent model for plant-fungal interactions2. It has been fully sequenced, and the genetics of its infective cycle, particularly the early events, have been established3,4. The life cycle begins with a spore germinating on a leaf surface, forming the specialized penetration structure called the appressorium. The appressorium penetrates the leaf tissue, and infection continues with the development of lesions which start the process of sporulation and spread disease4. Preventing any of these early events would drastically inhibit this devastating disease. Consequently, most current research on blast disease has been focused on the early infection steps, from the germinated conidia forming an appressorium to the development of the invasive hyphae and the biotrophic interfacial complex (BIC)5.
The vast amount of research on blast disease has been conducted in rice, even though M. oryzae is a significant pathogen for a variety of crops, and newly evolved strains are emerging as a global threat to wheat6. While rice is one of the top three staple crops used to feed the population, along with wheat and corn, barley is the fourth cereal grain in terms of livestock feed and beer production7. As the craft beer industry grows, so does the economic value of barley. There are distinct advantages of using M. oryzae and barley as a pathosystem to study blast disease. First, there are strains of M. oryzae that infect only barley, as well as strains that can infect multiple grass species. For example, 4091-5-8 infects primarily only barley, while Guy11 and 70-15 can infect both barley and rice8. These strains are genetically similar, and the infection process is comparable9. Second, under standard laboratory and greenhouse conditions, barley is easier to grow, as it doesn&#39;t have the complicated requirements of rice (concise temperature control, high humidity, specific light spectra). There are also imaging challenges with rice due to the hydrophobicity of the leaf surface, which barley does not exhibit10.
This protocol presents a simple method for isolating and effectively utilizing barley leaf sheaths for microscopic analysis of multiple infection stages, using common laboratory supplies and a smartphone for data collection. This method for the barley leaf sheath assay is adaptable for labs across the world as it requires minimal supplies, and yet provides a clear picture of the microscopic interaction between the pathogen and the first few cells it infects. Whereas pathogenicity assays, such as a spray or droplet inoculation, can provide a macro view of the pathogen&#39;s ability to form lesions, this assay allows the researcher to visualize specific steps of early infection, from pre-penetration events to colonization of epidermal cells. Further, researchers can easily compare infection with the wild-type fungus to infection with a mutant reduced in virulence.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell phone&#160;</td>
			<td>Google&#160;</td>
			<td>Pixel 4A</td>
			<td>Any smartphone with a rear facing camera that can be mounted in an a holder will suffice.&#160;</td>
		</tr>
		<tr>
			<td>Cell phone Microscope adapter</td>
			<td>Vankey</td>
			<td>B01788LT3S</td>
			<td>https://www.amazon.com/Vankey-Cellphone-Telescope-Binocular-Microscope/dp/B01788LT3S/ref=sr_1_2_sspa?keywords=vankey+cellphone+telescope+adapter+mount&#38;qid=1662568182&#38;sprefix= 
			vankey+%2Caps%2C63&#38;sr=8-2 
			-spons&#38;psc=1&#38;spLa=ZW5jcnlwd 
			GVkUXVhbGlmaWVyPUFKNklBR 
			jlCREJaMEcmZW5jcnlwdGVkSWQ 
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			WN0aW9uPWNsaWNrUmVkaXJlY3 
			QmZG9Ob3RMb2dDbGljaz10cnVl</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>AmScope</td>
			<td>FM690TC</td>
			<td>40x&#8211;2500x Trinocular upright epi-fluorescence microscope</td>
		</tr>
		<tr>
			<td>Oatmeal old fashioned rolled oats</td>
			<td>Quaker</td>
			<td>N/A</td>
			<td>https://www.amazon.com/Quaker-Oats-Old-Fashioned-Pack/dp/B00IIVBNK4/ref=asc_df_B00IIVBNK4/?tag=hyprod-20&#38;linkCode=df0 
			&#38;hvadid=312253390021&#38;hvpos= 
			&#38;hvnetw=g&#38;hvrand=98212627704 
			6839544&#38;hvpone=&#38;hvptwo=&#38;hvq 
			mt=&#38;hvdev=c&#38;hvdvcmdl=&#38;hvlocint 
			=&#38;hvlocphy=9007494&#38;hvtargid 
			=pla-568492637928&#38;psc=1</td>
		</tr>
		<tr>
			<td>ProMix BX</td>
			<td>ProMix</td>
			<td>1038500RG</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular coverglass</td>
			<td>Corning</td>
			<td>CLS2975245</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides, microscope</td>
			<td>Sigma-Aldrich</td>
			<td>S8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage micrometer&#160;</td>
			<td>OMAX</td>
			<td>A36CALM7</td>
			<td>0.1 mm and 0.01 mm Microscope calibration slide</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma-Aldrich</td>
			<td>T6146</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing early infection sites of rice blast disease (magnaporthe oryzae) on barley (hordeum vulgare) using a basic microscope and a smartphone

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.

A depiction of the initial workflow for this technique is displayed in Figure 1. The sheaths were harvested from 14-day-old susceptible &#34;Lacey&#34; barley plants (H. vulgare). The conidia were harvested from 10-day-old sporulating M. oryzae OMA plates, with a conidial suspension prepared using sterile ddH2O for a final concentration of 5 x 104 spores per mL. The inoculum suspension was directly applied to the leaf sheaths, which were secured to ste...

Watch this Scientific Journal Video about Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone at JoVE.com

Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more ...

visualizing-early-infection-sites-rice-blast-disease-magnaporthe

Research

JoVE Journal

Developmental Biology

1.5K Views.  University of Delaware. This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

Video: Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...