Name: an efficient clearing protocol for the study of seed development in tomato (solanum lycopersicum l.)
Uploaded: 2022-09-07T14:15:00
Description: Watch this Scientific Journal Video about An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L. at JoVE.com

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...

<ol>
	<li>. FAOSTAT Available from: <a target="_blank" href="https://www.fao.org/faostat/en/#data/QCL">https://www.fao.org/faostat/en/#data/QCL</a> (2022)</li><li>Olmstead, R. G., Bohs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+summary+of+molecular+systematic+research+in+Solanaceae:+1982-2006.">A summary of molecular systematic research in Solanaceae: 1982-2006.</a> Acta Horticulturae. 745, 255-268 (2007).</li><li>Consortium, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tomato+genome+sequence+provides+insights+into+fleshy+fruit+evolution.">The tomato genome sequence provides insights into fleshy fruit evolution.</a> Nature. 485 (7400), 635-641 (2012).</li><li>Ebert, A. W., Chou, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tomato+collection+maintained+by+AVRDC+-+The+World+Vegetable+Center:+composition,+germplasm+dissemination+and+use+in+breeding.">The tomato collection maintained by AVRDC - The World Vegetable Center: composition, germplasm dissemination and use in breeding.</a> Acta Horticulturae. 1101, 169-176 (2015).</li><li>Hilhorst, H., Groot, S., Bino, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tomato+seed+as+a+model+system+to+study+seed+development+and+germination.">The tomato seed as a model system to study seed development and germination.</a> Acta Botanica Neerlandica. 47, 169-183 (1998).</li><li>Chaban, I. A., Gulevich, A. A., Kononenko, N. V., Khaliluev, M. R., Baranova, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+structural+details+of+tomato+seed+coat+formation:+A+different+functional+role+of+the+inner+and+outer+epidermises+in+unitegmic+ovule.">Morphological and structural details of tomato seed coat formation: A different functional role of the inner and outer epidermises in unitegmic ovule.</a> Plants-Basel. 11 (9), 1101 (2022).</li><li>Iwahori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+temperature+injuries+in+tomato.+V.+Fertilization+and+development+of+embryo+with+special+reference+to+the+abnormalities+caused+by+high+temperature.">High temperature injuries in tomato. V. Fertilization and development of embryo with special reference to the abnormalities caused by high temperature.</a> Journal of The Japanese Society for Horticultural Science. 35 (4), 379-386 (1966).</li><li>Xiao, H., 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=Integration+of+tomato+reproductive+developmental+landmarks+and+expression+profiles,+and+the+effect+of+SUN.+on+fruit+shape.">Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN. on fruit shape.</a> BMC Plant Biology. 9 (1), 49 (2009).</li><li>Karssen, C. M., Haigh, A. M., Toorn, P., Weges, R., Taylorson, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+mechanisms+involved+in+seed+priming.">Physiological mechanisms involved in seed priming.</a> Recent advances in the development and germination of seeds. NATO ASI Series. 187, (1989).</li><li>Nonogaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+dormancy+and+germination-emerging+mechanisms+and+new+hypotheses.">Seed dormancy and germination-emerging mechanisms and new hypotheses.</a> Frontiers in Plant Science. 5, 233 (2014).</li><li>Doll, N. M., Ingram, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo-endosperm+interactions.">Embryo-endosperm interactions.</a> Annual Review of Plant Biology. 73, 293-321 (2022).</li><li>Serrani, J. C., Fos, M., Atar&#233;s, A., Garc&#237;a-Mart&#237;nez, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+gibberellin+and+auxin+on+parthenocarpic+fruit+growth+induction+in+the+cv+micro-tom+of+tomato.">Effect of gibberellin and auxin on parthenocarpic fruit growth induction in the cv micro-tom of tomato.</a> Journal of Plant Growth Regulation. 26 (3), 211-221 (2007).</li><li>Yang, 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=A+regulatory+gene+induces+trichome+formation+and+embryo+lethality+in+tomato.">A regulatory gene induces trichome formation and embryo lethality in tomato.</a> Proceedings of the National Academy of Sciences USA. 108 (29), 11836-11841 (2011).</li><li>Goetz, S., 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=Role+of+cis-12-oxo-phytodienoic+acid+in+tomato+embryo+development.">Role of cis-12-oxo-phytodienoic acid in tomato embryo development.</a> Plant Physiology. 158 (4), 1715-1727 (2012).</li><li>Ko, H. Y., Ho, L. H., Neuhaus, H. E., Guo, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transporter+SlSWEET15+unloads+sucrose+from+phloem+and+seed+coat+for+fruit+and+seed+development+in+tomato.">Transporter SlSWEET15 unloads sucrose from phloem and seed coat for fruit and seed development in tomato.</a> Plant Physiology. 187 (4), 2230-2245 (2021).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+tissue+clearing.">Clarifying tissue clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Kurihara, D., Mizuta, Y., Sato, Y., Higashiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClearSee:+A+rapid+optical+clearing+reagent+for+whole-plant+fluorescence+imaging.">ClearSee: A rapid optical clearing reagent for whole-plant fluorescence imaging.</a> Development. 142 (23), 4168-4179 (2015).</li><li>Vieites-Prado, A., Renier, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+clearing+and+3D+imaging+in+developmental+biology.">Tissue clearing and 3D imaging in developmental biology.</a> Development. 148 (18), (2021).</li><li>Kwiatkowska, M., Kad&#322;uczka, D., W&#281;dzony, M., Dedicova, B., Grzebelus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+of+a+clearing+protocol+to+study+crassinucellate+ovules+of+the+sugar+beet+(Beta+vulgaris+L.,+Amaranthaceae).">Refinement of a clearing protocol to study crassinucellate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae).</a> Plant Methods. 15, 71 (2019).</li><li>Ponitka, A., &#346;lusarkiewicz-Jarzina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleared-ovule+technique+used+for+rapid+access+to+early+embryo+development+in+Secale+cereale+&#215;+Zea+mays+crosses.">Cleared-ovule technique used for rapid access to early embryo development in Secale cereale &#215; Zea mays crosses.</a> Acta Biologica Cracoviensia. Series Botanica. 46, 133-137 (2014).</li><li>Ceccato, 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=Maternal+control+of+PIN1+is+required+for+female+gametophyte+development+in+Arabidopsis.">Maternal control of PIN1 is required for female gametophyte development in Arabidopsis.</a> PLoS One. 8 (6), 66148 (2013).</li><li>Wilkinson, L. G., Tucker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimised+clearing+protocol+for+the+quantitative+assessment+of+sub-epidermal+ovule+tissues+within+whole+cereal+pistils.">An optimised clearing protocol for the quantitative assessment of sub-epidermal ovule tissues within whole cereal pistils.</a> Plant Methods. 13, 67 (2017).</li><li>Hedhly, A., Vogler, H., Eichenberger, C., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+clearing+and+staining+of+Arabidopsis+flower+organs+and+Siliques.">Whole-mount clearing and staining of Arabidopsis flower organs and Siliques.</a> Journal of Visualized Experiments. (134), e56441 (2018).</li><li>Creff, A., Brocard, L., Ingram, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanically+sensitive+cell+layer+regulates+the+physical+properties+of+the+Arabidopsis+seed+coat.">A mechanically sensitive cell layer regulates the physical properties of the Arabidopsis seed coat.</a> Nature Communications. 6, 6382 (2015).</li><li>Yang, T., 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+B3+domain-containing+transcription+factor+ZmABI19+coordinates+expression+of+key+factors+required+for+maize+seed+development+and+grain+filling.">The B3 domain-containing transcription factor ZmABI19 coordinates expression of key factors required for maize seed development and grain filling.</a> Plant Cell. 33 (1), 104-128 (2021).</li><li>Anderson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hoyer's+solution+as+a+rapid+permanent+mounting+medium+for+bryophytes.">Hoyer's solution as a rapid permanent mounting medium for bryophytes.</a> Bryologist. 57 (3), 242-244 (1954).</li><li>Herr, J. 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+new+clearing-squash+technique+for+the+study+of+ovule+development+in+angiosperms.">A new clearing-squash technique for the study of ovule development in angiosperms.</a> American Journal of Botany. 58 (8), 785-790 (1971).</li><li>Yadegari, 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=Cell+differentiation+and+morphogenesis+are+uncoupled+in+Arabidopsis+raspberry+embryos.">Cell differentiation and morphogenesis are uncoupled in Arabidopsis raspberry embryos.</a> The Plant Cell. 6 (12), 1713-1729 (1995).</li><li>Grini, P. E., Jurgens, G., Hulskamp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+and+endosperm+development+is+disrupted+in+the+female+gametophytic+capulet+mutants+of+Arabidopsis.">Embryo and endosperm development is disrupted in the female gametophytic capulet mutants of Arabidopsis.</a> Genetics. 162 (4), 1911-1925 (2002).</li><li>Kataoka, K., Uemachi, A., Yazawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+growth+and+pseudoembryo+development+affected+by+uniconazole,+an+inhibitor+of+gibberellin+biosynthesis,+in+pat-2+and+auxin-Induced+parthenocarpic+tomato+fruits.">Fruit growth and pseudoembryo development affected by uniconazole, an inhibitor of gibberellin biosynthesis, in pat-2 and auxin-Induced parthenocarpic tomato fruits.</a> Scientia Horticulturae. 98 (1), 9-16 (2003).</li><li>de Jong, M., Wolters-Arts, M., Feron, R., Mariani, C., Vriezen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Solanum+lycopersicum+auxin+response+factor+7+(SlARF7)+regulates+auxin+signaling+during+tomato+fruit+set+and+development.">The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development.</a> Plant Journal. 57 (1), 160-170 (2009).</li><li>Roth, M., Florez-Rueda, A. M., Paris, M., Stadler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild+tomato+endosperm+transcriptomes+reveal+common+roles+of+genomic+imprinting+in+both+nuclear+and+cellular+endosperm.">Wild tomato endosperm transcriptomes reveal common roles of genomic imprinting in both nuclear and cellular endosperm.</a> Plant Journal. 95 (6), 1084-1101 (2018).</li><li>Orsi, C. H., Tanksley, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+an+ABC+transporter+gene+associated+with+seed+size+evolution+in+tomato+species.">Natural variation in an ABC transporter gene associated with seed size evolution in tomato species.</a> PLoS Genetics. 5 (1), 1000347 (2009).</li><li>Herridge, R. P., Day, R. C., Baldwin, S., Macknight, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+seed+size+in+Arabidopsis+for+mutant+and+QTL+discovery.">Rapid analysis of seed size in Arabidopsis for mutant and QTL discovery.</a> Plant Methods. 7 (1), 3 (2011).</li><li>Xu, T. T., Ren, S. C., Song, X. F., Liu, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLE19+expressed+in+the+embryo+regulates+both+cotyledon+establishment+and+endosperm+development+in+Arabidopsis.">CLE19 expressed in the embryo regulates both cotyledon establishment and endosperm development in Arabidopsis.</a> Journal of Experimental Botany. 66 (17), 5217-5227 (2015).</li><li>Ghadiri Alamdari, N., Salmasi, S., Almasi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+seed+mucilage+as+a+new+source+of+biodegradable+film-forming+material:+effect+of+glycerol+and+cellulose+nanofibers+on+the+characteristics+of+resultant+films.">Tomato seed mucilage as a new source of biodegradable film-forming material: effect of glycerol and cellulose nanofibers on the characteristics of resultant films.</a> Food and Bioprocess Technology. 14 (12), 2380-2400 (2021).</li><li>Gardner, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+botanical+clearing+technique.">An overview of botanical clearing technique.</a> Stain Technology. 50 (2), 99-105 (1975).</li><li>Beresniewicz, M. M., Taylor, A. G., Goffinet, M. C., Terhune, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+location+of+a+semipermeable+layer+in+seed+coats+of+leek+and+onion+(Liliaceae),+tomato+and+pepper+(Solanaceae).">Characterization and location of a semipermeable layer in seed coats of leek and onion (Liliaceae), tomato and pepper (Solanaceae).</a> Seed Science and Technology. 23 (1), 123-134 (1995).</li><li>Stebbins, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bleaching+and+clearing+method+for+plant+tissues.">A bleaching and clearing method for plant tissues.</a> Science. 87 (2245), 21-22 (1938).</li><li>Debenham, E. 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+modified+technique+for+the+microscopic+examination+of+the+xylem+of+whole+plants+or+plant+organs.">A modified technique for the microscopic examination of the xylem of whole plants or plant organs.</a> Annals of Botany. 3 (2), 369-373 (1939).</li><li>Morley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+clearing+of+plant+leaves+by+NaOH+in+association+with+oxygen.">Accelerated clearing of plant leaves by NaOH in association with oxygen.</a> Stain Technology. 43 (6), 315-319 (1968).</li></ol>

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 ...

This method can help researches quickly and effortlessly find abnormal periods of tomato embryo development. The main advantage of this protocol is a treatment of tomato seeds with sodium hypochlorite which provides a basis for the observation of tomato embryos. Begin with the chemical preparation and harvesting of the Solanum lycopersicum fruits three to 23 days after flowering or DAF.Immediately put the fruits on ice and divide them into early, middle and late fruits, based on the days after flowering. Break the early fruit, put it on a slide and collect fresh seeds carefully with precision forceps under 1x to 4x magnification of the stereo microscope. For middle and late fruits, break the fruit and directly collect fresh seeds using precision forceps.In the conventional method, transfer the freshly collected seeds immediately into a two milliliter centrifuge tube containing 1.5 milliliters FAA fixative and place the tube on the orbital shaker at 5 rpm for four hours at room temperature. Then dehydrate the seeds in 70%95%and 100%ethanol for one hour each on an orbital shaker at 5 rpm. Once done, place the seed in the three to five drops of clearing solution present on the slide, and gently cover it with a cover slip.Use a single concave slide for middle and late seeds. Depending on the developmental stages of the seeds, incubate them at room temperature for clearing. After incubation, observe the samples with a differential interference contrast, or 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. Transfer the fresh seeds collected from the broken fruits into a two milliliter centrifuge tube containing 1.5 milliliters of disinfectant solution. Incubate the samples on an orbital shaker at 30 rpm for three to 50 minutes at room temperature.Once the innermost seed coat outline is clearly visible, discard the disinfectant solution. For middle and late seeds, transfer the seeds onto a slide. Use forceps and a dissecting needle to remove the seed mucilage under 1x to 4x magnification of the stereo microscope.Transfer the seeds back into the original centrifuge tube using forceps. Wash them five times in 1.5 milliliters of deionized water for 10 seconds each, and discard the deionized water. Next, add the double volume of the clearing solution to the seeds.Depending on the seed stage, give the intermittent vacuum treatment for zero to 50 minutes, with 10 minute intervals after each vacuum treatment of 10 minutes. After vacuum treatment, replace the clearing solution. To facilitate the clearing of seeds, place the centrifuge tube protected from light for 30 minutes to seven days at room temperature.In the case of late embryos, replace the solution daily with a fresh clearing solution, followed by subjecting the seeds to vacuum treatment for 10 minutes. Place the cleared seeds on the slide or single concave slide. Using the DIC microscope equipped with a digital camera, adjust and optimize the transmitted light brightness, DIC slider and condenser aperture in real time for each sample and capture the image.In the conventional method of clearing S.lycopersicum seeds, the dense endosperm cells blocked the visualization of early embryos at 3 and 6 DAF. The decreased permeability during the seed growth resulted in fuzzy images after 9 DAF. The seed mucilage and seed coat gradually became denser, preventing the penetration of clearing agents from 13 DAF onward.The outline of the embryo inside 14 to 19 DAF seeds was extremely blurred, Despite the extended treatment time of seven days. The internal structure in 22 DAF seeds was completely invisible. The stripped seed coat mucilage produced by the seed coat epidermal cells was prominent from 13 DAF onward.The sodium hypochlorite treatment enabled a clear identification of the inner seed coat and detachment of most of the adherent mucilage without seed damage. In the optimized clearing protocol the seeds showed satisfactory transparency in all tested developmental stages. The distinct cell layers of the seed coat at 3 DAF, distinguishable endosperm cells at 5 DAF, the stick shaped embryo at seven DAF, the globular stage at 9 DAF, and the heart stage at 11 DAF were observed.In the optimized protocol, the degree of the curl of cotyledons and shoot apical meristem was very easily captured at various stages of embryonic development. Sterilization removes seed mucilage and seed coat pigment, which enhance seed penetration and visualization. Vacuum treatment can accelerate clearing solution penetration.

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Research

JoVE Journal

Developmental Biology

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 ...

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.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...