CSH protocols最新文献_第8页

Preparing recombinant gonad organ cultures. 制备重组性腺器官培养物。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5078

Blanche Capel, Jordan Batchvarov

引用次数: 7

Sex chromatin staining in amnion cells. 羊膜细胞的性染色质染色。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5079

Blanche Capel, Jordan Batchvarov

引用次数: 2

Astyanax mexicanus, the Blind Mexican Cave Fish: A Model for Studies in Development and Morphology. 盲墨西哥洞穴鱼:发育和形态研究的一个模型。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.emo107

Richard Borowsky

{"title":"Astyanax mexicanus, the Blind Mexican Cave Fish: A Model for Studies in Development and Morphology.","authors":"Richard Borowsky","doi":"10.1101/pdb.emo107","DOIUrl":"https://doi.org/10.1101/pdb.emo107","url":null,"abstract":"INTRODUCTIONThe perpetual darkness of caves has two important consequences for permanent inhabitants. First, eyes and pigmentation lose their primary functions. Second, in the absence of photosynthesis, food is rare. For these reasons, cave-adapted species typically have reduced eyes and pigmentation and increased or more efficient metabolisms. Additionally, other senses are usually augmented to compensate for the loss of vision. Identifying the genetic bases underlying these phenotypic changes will enhance our understanding of the specific pathways involved in control of these phenotypes and, in general, the evolutionary process. Unfortunately, the genetics of most cave animals cannot be studied because they are not easily bred. Blind Mexican tetras, Astyanax mexicanus, are the valuable exception to this rule because fish from the various cave populations are fully interfertile with one another and with eyed sister forms still living in nearby surface streams. Hybrids between surface and cave forms permit genetic analysis of their differences, and study of the pure forms as well as of hybrids allows study of their developmental differences. Quantitative trait loci (QTL) analysis has already identified some specific genes responsible for differences between cave and surface forms as well as other likely candidates; more will be added in the future. This system is a valuable addition to the array of existing models for the study of developmental and evolutionary genetics because cave populations are repositories of numerous naturally occurring mutations affecting development of the eyes and other senses, pigmentation, bone structure, metamerism, and metabolism. These alleles have been prescreened by natural selection for high viability, which simplifies their study. In contrast, new alleles obtained through mutagenesis in other model species are typically burdened with lower viability.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":" ","pages":"pdb.emo107"},"PeriodicalIF":0.0,"publicationDate":"2008-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.emo107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29702122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 30

Mnemiopsis leidyi Spawning and Embryo Collection. 雷氏背虾的产卵和胚胎收集。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5085

Kevin Pang, Mark Q Martindale

{"title":"Mnemiopsis leidyi Spawning and Embryo Collection.","authors":"Kevin Pang, Mark Q Martindale","doi":"10.1101/pdb.prot5085","DOIUrl":"https://doi.org/10.1101/pdb.prot5085","url":null,"abstract":"INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Because of its large size, fecundity, abundance in coastal areas, and recent introduction to European waters, Mnemiopsis leidyi (commonly called \"sea walnuts\") is the most highly studied ctenophore. Under optimal conditions, these self-fertile hermaphrodites are capable of reproduction at 2 wk of age and can release up to 10,000 eggs per day. Adults can be maintained in large aquaria with gentle aeration as long as they are well fed and can be spawned daily; multiple generations can be raised in the laboratory. This protocol describes how to collect embryos from M. leidyi. Under natural conditions, spawning normally occurs ~8 h after sunset, such that eggs are released under the cover of darkness. Because spawning is triggered by the onset of darkness, keeping animals under an artificial light regimen in the laboratory can alter the time of spawning. This protocol is designed to induce animal spawning at approximately 11:00 a.m.; however, it can be adjusted for other times. The duration from spawning to hatching of larvae is 18-24 h.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":" ","pages":"pdb.prot5085"},"PeriodicalIF":0.0,"publicationDate":"2008-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5085","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29702082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 21

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 3: Isolating DNA Plasmids in a 96-Well Plate Format. 核酸可编程蛋白阵列(NAPPA)的构建3:在96孔板中分离DNA质粒。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5058

Andrew J Link, Joshua Labaer

{"title":"Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 3: Isolating DNA Plasmids in a 96-Well Plate Format.","authors":"Andrew J Link, Joshua Labaer","doi":"10.1101/pdb.prot5058","DOIUrl":"https://doi.org/10.1101/pdb.prot5058","url":null,"abstract":"INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes a method for isolating the plasmids in a 96-well format.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":" ","pages":"pdb.prot5058"},"PeriodicalIF":0.0,"publicationDate":"2008-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5058","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29702125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Ctenophore whole-mount antibody staining. 栉水母全载抗体染色。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5086

Kevin Pang, Mark Q Martindale

{"title":"Ctenophore whole-mount antibody staining.","authors":"Kevin Pang, Mark Q Martindale","doi":"10.1101/pdb.prot5086","DOIUrl":"https://doi.org/10.1101/pdb.prot5086","url":null,"abstract":"INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. Because of the ease of embryo collection, their size (up to 1 mm in some species), and their rapid development, ctenophores have been attractive animals for experimental embryologists. This protocol describes how to fix ctenophore embryos and their cydippid larvae for antibody staining. Once the samples have been fixed, tissues are incubated with an antibody to the epitope of interest. A secondary antibody conjugated to a fluorescent molecule then reveals the expression pattern of the epitope. Fluorescent microscopy is used to visualize and document the signal. The protocol also includes methods for staining or counterstaining with a fluorescent derivative of phalloidin, which reveals F-actin in muscles and cell borders. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":" ","pages":"pdb.prot5086"},"PeriodicalIF":0.0,"publicationDate":"2008-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5086","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Ctenophore tissue preparation and extraction of RNA. 栉水母组织的制备及RNA的提取。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5089

Kevin Pang, Mark Q Martindale

{"title":"Ctenophore tissue preparation and extraction of RNA.","authors":"Kevin Pang, Mark Q Martindale","doi":"10.1101/pdb.prot5089","DOIUrl":"https://doi.org/10.1101/pdb.prot5089","url":null,"abstract":"INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to isolate total RNA from ctenophore embryos and larvae. After the specimens are sorted, cleaned, and concentrated, they are placed into TRI Reagent, a solution containing phenol and guanidine thiocyanate that allows for the effective isolation of total RNA. The resulting RNA can be used for various applications (e.g., to generate cDNA for reverse transcriptase-polymerase chain reactions). Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":" ","pages":"pdb.prot5089"},"PeriodicalIF":0.0,"publicationDate":"2008-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5089","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Breeding Astyanax mexicanus through Natural Spawning. 通过自然产卵繁殖墨西哥Astyanax。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5091

Richard Borowsky

引用次数: 20

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 4: DNA Biotinylation, Precipitation, and Arraying of Samples. 核酸可编程蛋白阵列(NAPPA)的构建4:DNA生物素化、沉淀和样品排列。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5059

Andrew J Link, Joshua Labaer

{"title":"Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 4: DNA Biotinylation, Precipitation, and Arraying of Samples.","authors":"Andrew J Link, Joshua Labaer","doi":"10.1101/pdb.prot5059","DOIUrl":"https://doi.org/10.1101/pdb.prot5059","url":null,"abstract":"INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes DNA biotinylation, precipitation, and arraying in preparation for protein expression.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":" ","pages":"pdb.prot5059"},"PeriodicalIF":0.0,"publicationDate":"2008-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5059","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29702126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Ctenophore whole-mount in situ hybridization. 栉水母整片原位杂交。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.prot5087

Kevin Pang, Mark Q Martindale

{"title":"Ctenophore whole-mount in situ hybridization.","authors":"Kevin Pang, Mark Q Martindale","doi":"10.1101/pdb.prot5087","DOIUrl":"https://doi.org/10.1101/pdb.prot5087","url":null,"abstract":"INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to fix, prepare, and hybridize antisense RNA probes in ctenophore embryos and cydippid larvae, as well as how to detect the probes using an alkaline phosphatase-conjugated antibody and colorimetric substrates. Using these techniques, it is possible to determine which cells or tissues express the gene of interest. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":" ","pages":"pdb.prot5087"},"PeriodicalIF":0.0,"publicationDate":"2008-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5087","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701401","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8