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https://www.readbyqxmd.com/read/29916178/quantification-of-neurotransmitters-from-intact-and-regenerating-planarians-using-uhplc-ms-srm-method
#1
Kannan Rangiah, Dasaradhi Palakodeti
Freshwater planarian species S. mediterranea is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. It is one of the best model systems available to address the basic biological mechanisms in the regeneration processes. Absolute quantification of metabolites from planarians is imperative to understand their role in the regeneration processes. Here we describe a stable isotope dilution ultrahigh performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC-MS/SRM) assay for a sensitive and quantitative assessment of neurotransmitters (NTs) in planaria...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916177/tissue-extracts-for-quantitative-mass-spectrometry-of-planarian-proteins-using-silac
#2
Alexander Böser, Hannes C A Drexler, Kerstin Bartscherer
SILAC (stable isotope labeling by amino acids in cell culture) proteomics enables the relative quantification of proteins in one or more biological samples by mass spectrometry. This technology is based on the metabolic incorporation of heavy isotope-labeled essential amino acids into nascent proteins in vivo. Here, we describe the preparation of SILAC protein samples from planarians, flatworms with high regenerative potential and tissue plasticity. Applications for SILAC proteomics of planarians include the analysis of protein abundances, protein-protein interactions and turnover rates during stem cell-based regeneration and tissue homeostasis...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916176/processing-schmidtea-mediterranea-for-transmission-electron-microscopy-classical-and-microwave-techniques
#3
John L Brubacher, Ana P Vieira, Juliette Azimzadeh
The flatworm Schmidtea mediterranea and related species are emerging model systems in such fields as stem-cell biology, regeneration, and evolutionary biology. Excellent molecular tools have been developed for S. mediterranea, but ultrastructural techniques have received less attention, which is unfortunate, as these methods are necessary to better understand the actual histological, cellular and subcellular features of regeneration and development. Tissue-processing regimens can be quite idiosyncratic for particular species or specimen types-what works for mammalian tissues or cell cultures will not necessarily give good results with freshwater planarians...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916175/live-imaging-of-planaria
#4
Wei Shen, Yun Shen, Yun Wah Lam, Danny Chan
Planarian regeneration involves a complex series of cellular events, precisely choreographed in space and time. Time-lapse imaging can provide powerful insights into tissue dynamics, as variously demonstrated in other model systems. However, time-lapse imaging of planarians has proven to be a challenge. Especially the requisite immobilization of the animals over extended periods of time is difficult, owing to their photophobic behavior and soft body architecture. Here, we describe a new embedding method using 2% (w/v) low melting agarose, and demonstrate that this method can effectively immobilize animals as long as 7 days...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916174/tissue-transplantations-in-planarians
#5
José I Rojo-Laguna, Emili Saló
Tissue transplantation is an important tool for in vivo studies of pattern organization and axis establishment or maintenance in planarians. Further, transplants can inform on cell movements under different regenerative conditions or genetic backgrounds. Here, we detail classical grafting strategies that have been variously employed in planarian research over the last few decades. These protocols remain similarly useful for addressing current and future questions in the field and the step-by-step protocol that we provide allows facile adaptation of this important method into the experimental repertoire of a laboratory...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916173/clonal-analysis-of-planarian-stem-cells-by-subtotal-irradiation-and-single-cell-transplantation
#6
Irving E Wang, Daniel E Wagner, Peter W Reddien
Stem cells, which both self-renew and produce differentiated progeny, represent fundamental biological units for the development, growth, maintenance, and regeneration of adult tissues. Characterization of stem cell lineage potential can be accomplished with clonal assays that interrogate stem cell output at the single-cell level. Here we present two methods for clonal analysis of individual proliferative cells (neoblasts) in the planarian Schmidtea mediterranea. The first method utilizes "subtotal" gamma irradiation to study rare surviving neoblasts and their clonal descendants in their native environment...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916172/a-subtractive-facs-method-for-isolation-of-planarian-stem-cells-and-neural-cells
#7
Tetsutaro Hayashi, Kiyokazu Agata
Planarians are among the metazoan organisms with the greatest regenerative abilities. This ability is based on their pluripotent stem cells, called neoblasts, which constitute 10-20% of the cells in their body. Elucidating the molecular mechanisms of the planarian stem cell system, for example, the maintenance of stem cell homeostasis and orchestration of lineage choices, contributes powerfully to the advancement of regenerative biology. Our group has developed fluorescence activated cell sorting (FACS) methodologies for the reliable isolation of planarian stem cells, which constitutes an important experimental asset in the field...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916171/rna-interference-in-planarians-feeding-and-injection-of-synthetic-dsrna
#8
Norito Shibata, Kiyokazu Agata
RNA interference (RNAi) is one of the simplest and easiest methods for specifically perturbing gene function in an organism. In planarian research, RNAi is one of the essential methods for defining gene functions not only during regeneration, but also during other life history stages. Since the first report of the efficacy of RNAi in planarians in 1999, several RNAi protocols have been reported. Here, we describe protocols to synthesize and deliver synthetic double-stranded RNA (dsRNA) to planarians, either by injection or by feeding...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916170/systemic-rna-interference-in-planarians-by-feeding-of-dsrna-containing-bacteria
#9
Carolyn E Adler, Alejandro Sánchez Alvarado
RNA interference (RNAi) is currently the only method available in planaria for assessing the function of particular genes. We describe here a method for performing body-wide gene knockdown, relying on dsRNA production in bacteria and subsequent delivery to planaria by feeding a liver-bacteria mixture. This method is ideal for screening many genes in parallel, in a cost-effective and reliable manner. We also describe a ligation-independent cloning strategy, which is used to rapidly transfer single genes into an RNAi vector that is also appropriate for downstream applications such as in situ hybridizations...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916169/detection-of-apoptotic-cells-in-planarians-by-whole-mount-tunel
#10
Brad Stubenhaus, Jason Pellettieri
Planarians have gained a well-deserved reputation as an excellent model organism for research on the biology of adult stem cells and their role in regeneration. Though less widely recognized, these animals also offer many advantages for investigating mechanisms and functions of programmed cell death in self-renewing tissues. Apoptosis complements stem cell division during physiological cell turnover and constitutes a prominent feature of the tissue remodeling process that restores anatomical scale and proportion during regeneration...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916168/whole-mount-brdu-staining-with-fluorescence-in-situ-hybridization-in-planarians
#11
Li-Chun Cheng, Alejandro Sánchez Alvarado
Detection of cell proliferation based on the incorporation of 5'-bromo-2'-deoxyuridine (BrdU) has become a standard approach for studying stem cell and progenitor cell populations in developing and adult tissue. In this chapter, we describe three BrdU administration methods for planarians and a staining protocol combining BrdU detection with whole-mount fluorescence in situ hybridization (FISH). Collectively, these protocols enable the combined analysis of BrdU-incorporation and endogenous gene expression, as for example during lineage tracing applications...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916167/general-principles-of-planarian-embryogenesis-and-its-analysis-by-in-situ-hybridization-and-immunohistochemistry-methods
#12
José María Martín-Durán
Thanks to their ability to regrow any missing body part after injury, planarians have become a well-established invertebrate model system in regenerative studies. However, planarians are also unique in their embryonic development, displaying ectolecithality, or the accumulation of embryonic nutrients into accessory cells accompanying the zygotes. Gaining a better understanding of their peculiar embryogenesis can offer answers to some fundamental questions regarding the appearance and evolution of planarian regenerative capacities, and in a broader context, the diversification of embryonic and postembryonic development in animals...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916166/rna-in-situ-hybridization-on-planarian-paraffin-sections
#13
Jordi Solana
RNA in situ hybridization techniques are an important tool for the study of gene expression patterns in freshwater planarians. Here I describe a RNA in situ hybridization method on histological paraffin sections of planarian tissue. This protocol allows the visualization of gene expression at cellular or subcellular resolution. Following paraffin-embedding and sectioning of planarians, the resulting sections are hybridized with hapten-labeled RNA probes. Subsequent immunological probe detection is carried out with either chromogenic or fluorescent development...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916165/whole-mount-in-situ-hybridization-of-planarians
#14
Ryan S King, Phillip A Newmark
Whole-mount in situ hybridization (WISH) and fluorescent whole-mount in situ hybridization (FISH) allow for visualization of specific mRNA transcripts to answer diverse biological questions. In planarians, in situ hybridization enables determination of gene expression profiles and identification of cell-type specific markers for analyzing experimental treatments. Here, we describe a robust whole-mount protocol for detecting gene expression patterns in the planarian Schmidtea mediterranea.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916164/immunohistochemistry-on-paraffin-embedded-planarian-tissue-sections
#15
Teresa Adell, Sara Barberán, Miquel Sureda-Gómez, María Almuedo-Castillo, Nidia de Sousa, Francesc Cebrià
Planarians are flatworms with almost unlimited regenerative abilities, which make them an excellent model for stem cell-based regeneration. To study the process of regeneration at the cellular level, immunohistochemical staining methods are an important tool, and the availability of such protocols is one of the prerequisites for mechanistic experiments in any animal model. Here, we detail protocols for paraffin embedding and immunostaining of paraffin sections of the model species Schmidtea mediterranea. This protocol yields robust results with a variety of commercially available antibodies...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916163/fixation-processing-and-immunofluorescent-labeling-of-whole-mount-planarians
#16
David J Forsthoefel, Kelly G Ross, Phillip A Newmark, Ricardo M Zayas
Efforts to elucidate mechanisms of regeneration in the planarian Schmidtea mediterranea have included the application of immunocytochemical methods to detect specific molecules and label cells and tissues in situ. Here we describe methods for immunofluorescent labeling of whole mount planarians. We outline protocols for fixation and steps for processing animals prior to immunolabeling, incorporating commonly utilized reagents for mucus removal, pigment bleaching, tissue permeabilization, and antigen retrieval...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916162/basic-histological-techniques-for-planarians
#17
Leigh Winsor, Ronald Sluys
The chapter provides a concise account of collecting techniques, and basic histological techniques for investigating planarians, intended for use by practicing scientists and postgraduate students new to the field. Selected hands-on methods are described that will lead to well-stained preparations suitable for light microscopic examination for subsequent taxonomic identification or general investigations into planarian functional cytology and histology. Major equipment items that are minimal essential requirements of a functional histological laboratory are indicated...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916161/planarian-high-molecular-weight-dna-isolation-by-spooling
#18
Shasha Zhang, Alejandro Sánchez Alvarado
High-quality large molecular weight genomic DNA is important for genomic studies. Most commercial available genomic DNA purification kits have failed to generate high molecular weight DNA of sufficient quality from planarians. Here, we describe a simple and efficient genomic DNA isolation method, which has worked for several different planarian species, including Schmidtea mediterranea. This phenol-chloroform based method can be used to obtain genomic DNA of up to 150 kb and can be used for bacterial artificial chromosome (BAC) library construction, next-generation sequencing and PCR cloning...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916160/small-and-large-scale-high-molecular-weight-genomic-dna-extraction-from-planarians
#19
Markus A Grohme, Miquel Vila-Farré, Jochen C Rink
High-quality genomic DNA extraction is a starting point for many downstream applications in modern molecular biology. Here, we describe a simple method for isolating high molecular weight genomic DNA from planarians. The method is based on tissue lysis by a mixture of a chaotropic salt and detergent followed by organic extraction to remove proteins and lipids followed by a postpurification step to remove contaminating polysaccharides. The isolated DNA is of high molecular weight and compatible with polymerase chain reaction, cloning, or next-generation sequencing library preparation...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29916159/total-rna-isolation-from-planarian-tissues
#20
Shang-Yun Liu, Jochen C Rink
Isolation of high-quality and nondegraded RNA is a prerequisite for many modern molecular biology applications. A variety of RNA extraction products and protocols are available for the standard model organisms, yet species-specific peculiarities of less well studied organisms often require specific protocol adaptations. Here we describe a robust RNA extraction protocol for planarians that is widely used in the community. The protocol combines tissue homogenization in TRIzol with phenol-chloroform extraction and subsequent purification over commercial columns...
2018: Methods in Molecular Biology
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