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Recording from Drosophila Larval Body Wall Muscles: Passive Membrane Properties and Basic Features of Synaptic Transmission. 果蝇幼虫体壁肌肉的记录：被动膜特性和突触传递的基本特征。
Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108131
Bing Zhang, Bryan Stewart
{"title":"Recording from <i>Drosophila</i> Larval Body Wall Muscles: Passive Membrane Properties and Basic Features of Synaptic Transmission.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108131","DOIUrl":"10.1101/pdb.prot108131","url":null,"abstract":"<p><p>The <i>Drosophila</i> larval body wall muscle preparation was first used for electrophysiological analysis in the 1970s. This preparation has become the \"gold standard\" for studying neuronal excitability as well as synaptic transmission. Here, we first describe the steps for performing intracellular recording from fly larval body wall muscles and then explain how to record and analyze spontaneous and evoked synaptic potentials. Methods used include larval dissection (filleting), identification of muscle fibers and their innervating nerves, the use of the micromanipulator and microelectrode in penetrating the muscle membrane, and nerve stimulation to evoke synaptic potentials.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108131"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189536","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: 0
Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Larval Neuromuscular Junction. 果蝇幼虫神经肌肉接头处单个突触电流的聚焦记录
Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108133
Bing Zhang, Bryan Stewart
{"title":"Focal Recording of Synaptic Currents from Single Boutons at the <i>Drosophila</i> Larval Neuromuscular Junction.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108133","DOIUrl":"10.1101/pdb.prot108133","url":null,"abstract":"<p><p>Focal recording is an extracellular method for studying synaptic transmission at the <i>Drosophila</i> larval neuromuscular junction (NMJ) designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all the boutons as occurs with intracellular recording or two-electrode voltage-clamp. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons. A compound microscope with epifluorescent imaging capability is very helpful but not essential; any GFP <i>Drosophila</i> strain that labels the nerve terminal or synaptic boutons can be used to locate the boutons. A particularly useful strain is Mhc-CD8-Sh-GFP, containing a GFP molecule that is expressed in muscle, localizes to the postsynaptic apparatus, and outlines boutons. Vital fluorescent dyes (such as 4-Di-2-Asp) may also be applied to the dissected preparation to help locate boutons. The microscope should be equipped for differential interference contrast (DIC or Nomarski) optics if fluorescence is not used.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108133"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189535","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: 0
Tools and Resources at the Maize Genetics and Genomics Database (MaizeGDB). 玉米遗传学和基因组学数据库（MaizeGDB）的工具和资源。
Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.over108430
Margaret R Woodhouse, Ethalinda K Cannon, John L Portwood, Jack M Gardiner, Rita K Hayford, Olivia Haley, Carson M Andorf
{"title":"Tools and Resources at the Maize Genetics and Genomics Database (MaizeGDB).","authors":"Margaret R Woodhouse, Ethalinda K Cannon, John L Portwood, Jack M Gardiner, Rita K Hayford, Olivia Haley, Carson M Andorf","doi":"10.1101/pdb.over108430","DOIUrl":"10.1101/pdb.over108430","url":null,"abstract":"<p><p>The Maize Genetics and Genomics Database (MaizeGDB) is the community resource for maize researchers, offering a suite of tools, informatics resources, and curated data sets to support maize genetics, genomics, and breeding research. Here, we provide an overview of the key resources available at MaizeGDB, including maize genomes, comparative genomics, and pan-genomics tools. This review aims to familiarize users with the range of options available for maize research and highlights the importance of MaizeGDB as a central hub for the maize research community. By providing a detailed snapshot of the database's capabilities, we hope to enable researchers to make use of MaizeGDB's resources, ultimately assisting them to better study the evolution and diversity of maize.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.over108430"},"PeriodicalIF":0.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141995470","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: 0
Performing Quantitative PCR after Chromatin Immunoprecipitation (ChIP) of Drosophila Antennal and Brain Samples. 在果蝇触角和大脑样本的染色质免疫沉淀 (ChIP) 之后进行定量 PCR。
Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108143
Chengcheng Du, Pelin Volkan
{"title":"Performing Quantitative PCR after Chromatin Immunoprecipitation (ChIP) of <i>Drosophila</i> Antennal and Brain Samples.","authors":"Chengcheng Du, Pelin Volkan","doi":"10.1101/pdb.prot108143","DOIUrl":"10.1101/pdb.prot108143","url":null,"abstract":"<p><p>Chromatin immunoprecipitation (ChIP) is a technique used to study specific protein-DNA interaction. Briefly, in this technique, antibodies to proteins of interest are used to isolate regions of DNA where these proteins bind. ChIP samples can be processed and analyzed in different ways. One of the approaches for assessing the results of ChIP experiments is quantitative PCR (qPCR). qPCR is used to quantitatively measure the amount of DNA fragments that have been isolated, reflecting the signal of specific proteins interacting with these fragments. This protocol describes both the \"percent input\" method and the \"fold enrichment\" method for ChIP-qPCR data analysis using <i>Drosophila</i> tissues as an example. The \"percent input\" method measures signals of DNA fragments against the input measurement. In contrast, the \"fold enrichment\" method quantifies the amplified signal strength relative to a background control. Because the quality of primers is critical for the reliability of ChIP-qPCR results, this protocol also describes how to measure primer amplification efficiency using <i>Drosophila</i> genomic DNA.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108143"},"PeriodicalIF":0.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140058828","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: 0
Clonal Variant Analysis of Antibody Engineering Libraries. 抗体工程库的克隆变异分析。
Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108626
Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky
{"title":"Clonal Variant Analysis of Antibody Engineering Libraries.","authors":"Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky","doi":"10.1101/pdb.prot108626","DOIUrl":"10.1101/pdb.prot108626","url":null,"abstract":"<p><p>In vitro antibody evolution is a powerful technique for improving monoclonal antibodies. This can be achieved by generating artificial diversity on an antibody template, which can be done using different in vitro diversification techniques. The resulting libraries consist of single- or multimutant variants of a defined antibody template that are screened for improved function using antibody display. Here, we describe a bioinformatic protocol for tracking synthetic antibody variants using high-throughput sequencing across screening rounds, enabling efficient high-throughput interpretation of the function of individual mutations in sorted antibody display libraries. The protocol enables a user to achieve precision analysis and interpretation of clonal antibody variant data for discovery purposes, especially for high-throughput antibody engineering or optimization against target antigens.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108626"},"PeriodicalIF":0.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142715588","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: 0
Clonal Lineage and Gene Diversity Analysis of Paired Antibody Heavy and Light Chains. 成对抗体重链和轻链的克隆谱系和基因多样性分析
Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108628
Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky
{"title":"Clonal Lineage and Gene Diversity Analysis of Paired Antibody Heavy and Light Chains.","authors":"Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky","doi":"10.1101/pdb.prot108628","DOIUrl":"10.1101/pdb.prot108628","url":null,"abstract":"<p><p>Antibodies consist of unique variable heavy (V<sub>H</sub>) and variable light (V<sub>L</sub>) chains, and both are required to fully characterize an antibody. Methods to detect paired heavy and light chain variable regions (V<sub>H</sub>:V<sub>L</sub>) using high-throughput sequencing (HTS) have recently enabled large-scale analysis of complete functional antibody responses. Here, we describe an HTS computational pipeline to analyze paired V<sub>H</sub>:V<sub>L</sub> antibody sequences and obtain a comprehensive profile of immune diversity landscapes, including gene usage, antibody isotypes, and clonal lineage analysis. This protocol uses Illumina MiSeq 2 × 300-bp sequencing data and integrates with several different computational tools for flexible analyses of paired V<sub>H</sub>:V<sub>L</sub> gene repertoire data to enable efficient antibody discovery.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108628"},"PeriodicalIF":0.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142715587","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: 0
Antibody Data Analysis from Diverse Immune Libraries. 来自不同免疫库的抗体数据分析
Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108627
Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky
{"title":"Antibody Data Analysis from Diverse Immune Libraries.","authors":"Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky","doi":"10.1101/pdb.prot108627","DOIUrl":"10.1101/pdb.prot108627","url":null,"abstract":"<p><p>Antibody functional screening studies and next-generation sequencing require careful processing and interpretation of sequence data for optimal results. Here, we provide a detailed protocol for the functional analysis of antibody gene data, including antibody repertoire quantification and functional mapping of high-throughput screening data based on enrichment ratio values, which are a simple way to determine the enrichment of each sequenced antibody after sorting a display library against desired antigens. This protocol enables a user to apply a set of simple yet powerful bioinformatic tools for high-throughput analysis and interpretation of antibody data that is especially well suited for display library screening and for antibody discovery applications.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108627"},"PeriodicalIF":0.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142715585","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: 0
Sample Preparation for Chromatin Immunoprecipitation (ChIP) from Drosophila Antennal and Brain Samples. 果蝇触角和大脑样本的染色质免疫沉淀 (ChIP) 样品制备。
Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108140
Chengcheng Du, Pelin Volkan
{"title":"Sample Preparation for Chromatin Immunoprecipitation (ChIP) from <i>Drosophila</i> Antennal and Brain Samples.","authors":"Chengcheng Du, Pelin Volkan","doi":"10.1101/pdb.prot108140","DOIUrl":"10.1101/pdb.prot108140","url":null,"abstract":"<p><p>Chromatin immunoprecipitation (ChIP) is a well-characterized procedure used to reveal specific patterns of protein-DNA interactions and identify the binding sites of proteins on DNA. ChIP has been used to study many aspects of <i>Drosophila</i> biology, including neurobiology. This protocol describes in detail how to prepare cross-linked chromatin from <i>Drosophila</i> antennae and brains followed by immunoprecipitation (X-ChIP). We first describe tissue dissection, chromatin cross-linking with formaldehyde, quenching of the cross-linking, homogenization of tissues, and sonication for shearing the chromatin. Additionally, we describe how to optimize the sonication efficiency and fixation time and concentration using <i>Drosophila</i> brain samples as an example. These parameters are crucial for successful ChIP.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108140"},"PeriodicalIF":0.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140058829","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: 0
Beyond Single Clones: High-Throughput Sequencing in Antibody Discovery. 超越单克隆：抗体发现中的高通量测序。
Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.top107772
Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky
{"title":"Beyond Single Clones: High-Throughput Sequencing in Antibody Discovery.","authors":"Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky","doi":"10.1101/pdb.top107772","DOIUrl":"10.1101/pdb.top107772","url":null,"abstract":"<p><p>Antibody repertoire sequencing and display library screening are powerful approaches for antibody discovery and engineering that can connect DNA sequence with antibody function. Antibody display and screening studies have made a tremendous impact on immunology and biotechnology over the last decade, accelerated by technological advances in high-throughput DNA sequencing techniques. Indeed, bioinformatic analysis of antibody DNA library data has now taken a central role in modern antibody drug discovery, and is also critical for many ongoing studies of human immune development. Here, we describe current trends in antibody DNA library screening and analysis, and introduce a selection of protocols describing fundamental bioinformatic techniques to enable scientists to efficiently study antibody DNA libraries.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top107772"},"PeriodicalIF":0.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142715586","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: 0
Chromatin Immunoprecipitation (ChIP) Using Drosophila Antennal and Brain Samples. 利用果蝇触角和大脑样本进行染色质免疫沉淀（ChIP）
Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108142
Chengcheng Du, Pelin Volkan
{"title":"Chromatin Immunoprecipitation (ChIP) Using <i>Drosophila</i> Antennal and Brain Samples.","authors":"Chengcheng Du, Pelin Volkan","doi":"10.1101/pdb.prot108142","DOIUrl":"10.1101/pdb.prot108142","url":null,"abstract":"<p><p>Chromatin immunoprecipitation (ChIP) is a common approach for studying the binding pattern of proteins on DNA sequences or the landscape of histones with different marks throughout the genome. ChIP is used on various organisms, including <i>Drosophila</i> This protocol provides a detailed overview of the immunoprecipitation portion of a ChIP procedure from samples of <i>Drosophila</i> nervous systems, specifically antennae and brains, that have already been fixed and sheared. These methods can be applied to other tissues of interest after optimizing for sample size and other relevant parameters.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108142"},"PeriodicalIF":0.0,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140058871","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: 0
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