The authors are grateful to Dr. Jie Le and Dr. Xiufen Song for their helpful suggestions on differential interference contrast microscopy and conventional clearing method, respectively. This research was financed by the National Natural Science Foundation of China (31870299) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences. Figure&#8197;2 was created with BioRender.com.

1. Preparation of solutions
<ol>
	<li>Prepare FAA fixative by adding 2.5 mL of 37% formaldehyde, 2.5 mL of glacial acetic acid, and 45 mL of 70% ethanol in a 50 mL centrifuge tube. Vortex and store it at 4 &#176;C. Freshly prepare FAA fixative just before use. 
	CAUTION: 37% formaldehyde is corrosive and potentially carcinogenic if exposed or inhaled. The fixative must be performed in a fume hood while wearing appropriate personal protective equipment.</li>
	<li>Prepare clearing solution by adding 5 mL of 100% glycerol, 40 g chloral hydrate, and 10 mL of distilled water in a 100 mL glass bottle wrapped with tin foil. Dissolve using a magnetic stirrer overnight at room temperature. The prepared solution can be stored at 4 &#176;C for up to 6 months. 
	CAUTION: Chloral hydrate is carcinogenic and has a pungent smell. Perform the experiment in a fume hood and wear appropriate personal protective equipment. Chloral hydrate is easy to deliquesce in the air and should not be stored in large quantities. The clearing solution can decompose when exposed to light and should be kept away from light.</li>
	<li>Prepare the disinfectant solution by adding 10 mL of 6% sodium hypochlorite, 40 mL distilled water, and 50 &#181;L Tween-20 in a 50 mL centrifuge tube. Vortex and store it at room temperature. Freshly prepare the disinfectant solution just before use. 
	NOTE:&#160;The effective chlorine content of sodium hypochlorite that has been placed for a long time may decrease, and the content of 6% sodium hypochlorite can be increased according to the actual situation.&#160;</li>
</ol>
2. Seed collection
<ol>
	<li>Sow tomato seeds (S. lycopersicum L. cv. Micro-Tom, see Table of Materials) in 8 cm &#215; 8 cm &#215; 8 cm square basins full with a 1:1 mixture of flower nutrient soil and substrate (v/v) (see Table of Materials), and grow in a growth room with 16/8 h light/dark periods at 24 &#177; 2 &#176;C (day) and 18 &#177; 2 &#176;C (night), 60%-70% relative humidity, and a light intensity of 4,000 Lux. Approximately 6 weeks later, plants entered the flowering stage.</li>
	<li>Tag the flowering date of independent flowers at anthesis (opening angle of the petals is 90&#176;), and record the day after flowering (DAF).</li>
	<li>Harvest the fruits from 3 to 23 DAF tomato plants and immediately put them on ice. Divide fruits (seeds or embryos) from 3 to 23 DAF into early (3-10 DAF), middle (11-16 DAF), and late (17-23 DAF) fruits (seeds or embryos). 
	NOTE: Do not collect too many fruits at a time and ensure that the seeds in each fruit are stripped within 1 h for further treatment.</li>
	<li>For early fruits, break the fruit and put it on a slide, and collect fresh seeds carefully with precision forceps under the stereomicroscope (see Table of Materials) at 1x to 4x magnification. For middle and late fruits, break the fruit and directly collect fresh seeds using precision forceps.</li>
</ol>
3. Chloral hydrate-based clearing of seeds
NOTE: Conventional35 and optimized protocols were compared in this study for their seed clearing efficiency.
<ol>
	<li>Clearing using a conventional protocol
	<ol>
		<li>Place fresh seeds (obtained in step 2.4) immediately in a 2 mL centrifuge tube containing 1.5 mL FAA fixative and incubate on an orbital shaker (5 rpm, see Table of Materials) for 4 h at room temperature.</li>
		<li>Dehydrate these seeds in an ethanol series of 70%, 95%, and 100% ethanol (v/v) for 1 h each on an orbital shaker (5 rpm).</li>
		<li>Place the seeds in 3-5 drops of clearing solution (~100 &#181;L) on the slide and gently cover the sample with a coverslip. Replace the slide with a single concave slide (see Table of Materials) for middle and late seeds.</li>
		<li>Keep these slides or single concave slides at room temperature for 30 min (3 DAF), 2 h (6 DAF), 1 day (9 DAF), 3 days (12 DAF), or 7 days (14 to 22 DAF) depending on the developmental stages of the seed material (the younger the materials, the faster the clearing speed).</li>
		<li>Observe the samples with a differential interference contrast (DIC) microscope equipped with a digital camera (see Table of Materials) at 10x, 20x, and 40x magnification. Adjust and optimize the transmitted light brightness, DIC slider, and condenser aperture in real time for each sample and capture images.</li>
	</ol>
	</li>
	<li>Clearing using the optimized protocol
	<ol>
		<li>Place fresh seeds (obtained in step 2.4) directly into a 2 mL centrifuge tube containing 1.5 mL disinfectant solution (step 1.3). 
		NOTE: The number of seeds collected in the 2 mL centrifuge tube varies according to the developmental stage of the seeds. Details are listed in Table 1.</li>
		<li>Incubate the samples on an orbital shaker (30 rpm) at room temperature for 3 to 50 min until the innermost seed coat outline is clearly visible. Discard the disinfectant solution. 
		NOTE: The incubation time required depends on the seed developmental stage (the later the seeds, the longer will be the incubation time). Details are listed in Table 1.</li>
		<li>For middle and late seeds, transfer the seeds onto a slide and remove the seed mucilage with forceps and a dissecting needle under a stereomicroscope at 1x to 4x magnification. Transfer the seeds back into the original centrifuge tube using forceps. 
		NOTE: This step is not necessary for early seeds.</li>
		<li>Wash the seeds 5x with 1.5 mL deionized water, 10 s each time. Discard the deionized water.</li>
		<li>Add a clearing solution of 2 &#215; the volume of the seeds, followed by vacuum treatment for 0 to 50 min (Table 1) with a vacuum pump (see Table of Materials). Intermittent vacuum treatment is performed with each vacuum treatment for 10 min spaced at 10 min intervals.</li>
		<li>Replace with a fresh clearing solution of 2 &#215; the volume of the seeds. Keep the 2 mL centrifuge tube containing the seeds at room temperature and protected from light for 30 min to 7 days to facilitate clearing (Table 1). During the incubation, for late embryos, replace the clearing solution daily with fresh solution and subject the seeds to vacuum treatment for 10 min.</li>
		<li>Prepare the cleared seeds on slides or single-concave slides and observe with the DIC microscope equipped with a digital camera at 10x, 20x, and 40x magnification. Adjust and optimize the transmitted light brightness, DIC slider, and condenser aperture in real time for each sample and capture images.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

Compared to mechanical sectioning, the clearing technology is more advantageous for three-dimensional imaging as it retains the integrity of plant tissues or organs16. Conventional clearing protocols are often limited to small samples due to easier penetration of chemical solutions. Tomato seed is a problematic sample for tissue clearing because it is about 70 times larger than an Arabidopsis seed in size and has more permeability barriers. The Arabidopsis seed coat is composed o...

Tomato (S. lycopersicum L.) is one of the most important vegetable crops around the world, with an output of 186.8 million tons of fleshy fruits from 5.1 million hectares in 20201. It belongs to the large Solanaceae family with about 2,716 species2, including many commercially important crops such as eggplant, peppers, potato, and tobacco. The cultivated tomato is a diploid species (2n = 2x = 24) with a genome size of approximately 900 Mb3. For a long time, great effort has been made toward tomato domestication and breeding by selecting desirable traits from wild Solanum spp. There are over 5,000 tomato accessions listed in the Tomato Genetics Resource Center and more than 80,000 germplasm of tomatoes are stored worldwide4. The tomato plant is perennial in the greenhouse and propagates by seeds. A mature tomato seed consists of three major compartments: a full-grown embryo, residual cellular-type endosperm, and a hard seed coat5,6 (Figure 1A). After double fertilization, the development of cellular-type endosperm precedes the development of zygotes. At ~5-6 days after flowering (DAF), two-celled proembryo is first observed when the endosperm consists of six to eight nuclei7. In Solanum pimpinellifolium, the embryo approaches its final size after 20 DAF, and seeds are viable for germination after 32 DAF8. As the embryo develops, the endosperm is gradually absorbed and only a small amount of endosperm remains in the seed. The residual endosperm consists of micropylar endosperm surrounding the radicle tip, and lateral endosperm in the rest of the seed9,10. The outer seed coat is developed from thickened and lignified outer epidermis of the integument, and with the dead layers of integument remnants, they form a hard shell to protect the embryo and endosperm5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64445/64445fig01.jpg" /> 
Figure 1: Schematic representation of a mature seed in Solanum lycopersicum and Arabidopsis thaliana. (A) Longitudinal anatomy of a mature tomato seed. (B) Longitudinal anatomy of a mature Arabidopsis seed. A mature tomato seed is approximately 70 times larger in size than an Arabidopsis seed. Scale bars = (A) 400 &#181;m, (B) 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64445/64445fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Production of high-quality tomato seeds depends on the coordination between the embryo, the endosperm, and the maternal seed components11. Dissecting key genes and networks in seed development requires a deep and full-track phenotypic recording of mutant seeds. Conventional embedding-sectioning techniques, such as the semi-thin section and paraffin section, are widely applied to tomato seeds to observe the local and finer structures of the embryo12,13,14,15. However, analyzing the seed development from thin sections is usually laborious and lacks z-axis spatial resolution. In comparison, tissue clearing is a fast and efficient method to pinpoint the developmental stage of embryo defects that are most likely to occur16. The clearing method reduces the opaqueness of internal tissue by homogenizing the refractive index with one or more biochemical agents16. Whole tissue clearing allows observation of a plant tissue structure without destroying its integrity, and the combination of clearing technology and three-dimensional imaging has become an ideal solution to obtain information on the morphology and developmental state of a plant organ17,18. Over the years, seed clearing techniques have been used in various plant species, including Arabidopsis thaliana, Hordeum vulgare, and Beta vulgaris19,20,21,22,23.&#160;Among these, the whole-mount ovule clearing technology has been an efficient approach to studying seed development of Arabidopsis, due to its small size, 4-5 layers of the seed coat cell, and the nuclear-type endosperm24,25. With the continuous updating of different clearing mixtures, such as the emergence of Hoyer&#39;s solution26, internal structures of the barley ovule were imaged with a high degree of clarity although its endosperm makes up the bulk of the seeds. Embryogenesis of sugar beet can be observed by clearing combined with vacuum treatment and softening with hydrochloric acid19. Nonetheless, unlike the species mentioned above, embryological observations by clearing protocols in tomato seeds have not been reported. This prevents detailed investigation into the embryonic and seed development of tomatoes.
Chloral hydrate is commonly used as a clearing solution that allows the immersed tissues and cells to be displayed on different optical planes, and substantially preserves the cells or tissue components27,28,29. Chloral hydrate-based clearing protocol has been successfully used for the whole-mount clearing of seeds to observe the embryo and endosperm of Arabidopsis21,28. However, this clearing solution is not efficient in clearing tomato seeds, which are more impermeable than Arabidopsis seeds. The physical barriers include: (1) the tomato integument has nearly 20 cell layers at 3 to 15 DAF30,31, (2) the tomato endosperm is cellular-type, not nuclear-type32, and (3) tomato seeds are about 70 times larger in size33,34 and (4) produce large amounts of seed coat mucilage, which blocks the penetration of clearing reagents and affects the visualization of embryo cells.
Therefore, this report presents an optimized chloral hydrate-based clearing method for whole-mount clearing of tomato seeds at different stages, which allows deep imaging of the embryo development process (Figure 2).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,000 &#181;L pipette</td>
			<td>GILSON</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 &#181;L pipette tips</td>
			<td>Corning</td>
			<td>T-1000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-200-C</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>DAMAO</td>
			<td>685-2013</td>
			<td></td>
		</tr>
		<tr>
			<td>5,000 &#181;L pipette</td>
			<td>Eppendorf</td>
			<td>3120000275</td>
			<td></td>
		</tr>
		<tr>
			<td>5,000 &#181;L pipette tips</td>
			<td>biosharp</td>
			<td>BS-5000-TL</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol</td>
			<td>BOYUAN</td>
			<td>678-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle glass</td>
			<td>Fisher</td>
			<td>FB800-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral Hydrate</td>
			<td>Meryer</td>
			<td>M13315-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Leica</td>
			<td>384200</td>
			<td></td>
		</tr>
		<tr>
			<td>DIC microscope</td>
			<td>Zeiss</td>
			<td>Axio Imager A1</td>
			<td>10x, 20x and 40x magnification</td>
		</tr>
		<tr>
			<td>Disinfectant</td>
			<td>QIKELONGAN</td>
			<td>17-9185</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>Bioroyee</td>
			<td>17-9140</td>
			<td></td>
		</tr>
		<tr>
			<td>Flower nutrient soil</td>
			<td>FANGJIE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>HAIOU</td>
			<td>4-94</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>BOYUAN</td>
			<td>676-2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Solarbio</td>
			<td>G8190</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RET basic</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Tom</td>
			<td>Tomato Genetics Resource Center</td>
			<td>LA3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>QILINBEIER</td>
			<td>QB-206</td>
			<td></td>
		</tr>
		<tr>
			<td>Seeding substrate</td>
			<td>PINDSTRUP</td>
			<td>LV713/018-LV252</td>
			<td>Screening:0-10 mm</td>
		</tr>
		<tr>
			<td>Single concave slide</td>
			<td>HUABODEYI</td>
			<td>HBDY1895</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Leica</td>
			<td>3800381</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>S8 APO</td>
			<td>1x to 4x magnification</td>
		</tr>
		<tr>
			<td>Tin foil</td>
			<td>ZAOWUFANG</td>
			<td>613</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>SHIDING</td>
			<td>SHB-III</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex meter</td>
			<td>Silogex</td>
			<td>MX-S</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient clearing protocol for the study of seed development in tomato (solanum lycopersicum l.)

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. Visualization of finer embryonic structure within a tomato seed is often hampered by seed coat mucilage, multi-cell-layered integument, and a thick-walled endosperm, which needs to be resolved by laborious embedding-sectioning. A simpler alternative is to employ tissue clearing techniques that turn the seed almost transparent using chemical agents. Although conventional clearing procedures allow deep insight into smaller seeds with a thinner seed coat, clearing tomato seeds continues to be technically challenging, especially in the late developmental stages.
Presented here is a rapid and labor-saving clearing protocol to observe tomato seed development from 3 to 23 days after flowering when embryonic morphology is nearly complete. This method combines chloral hydrate-based clearing solution widely used in Arabidopsis with other modifications, including the omission of Formalin-Aceto-Alcohol (FAA) fixation, the addition of sodium hypochlorite treatment of seeds, removal of the softened seed coat mucilage, and washing and vacuum treatment. This method can be applied for efficient clearing of tomato seeds at different developmental stages and is useful in full monitoring of the developmental process of mutant seeds with good spatial resolution. This clearing protocol may also be applied to deep imaging of other commercially important species in the Solanaceae.

When tomato seeds were cleared using a conventional method as in Arabidopsis, dense endosperm cells blocked the visualization of early tomato embryos at 3 DAF and 6 DAF (Figure 3A,B). As the total volume of the embryo increased, a globular embryo was barely distinguishable at 9 DAF (Figure 3C). However, as the seed size continued to increase, its permeability decreased, resulting in a fuzzy heart embryo at 12 DAF (F...

Watch this Scientific Journal Video about An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L. at JoVE.com

An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L.

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental ...

an-efficient-clearing-protocol-for-study-seed-development-tomato

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JoVE Journal

Developmental Biology

3.8K Views.  Chinese Academy of Sciences. The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

Video: An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

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 ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.