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Protocol for immunofluorescence detection and quantification of phosphorylated SMAD proteins in human blastocysts. 人胚泡中磷酸化SMAD蛋白的免疫荧光检测和定量方法。
IF 1.3
STAR Protocols Pub Date : 2025-05-28 DOI: 10.1016/j.xpro.2025.103849
Todd Fallesen, A Sophie Brumm
{"title":"Protocol for immunofluorescence detection and quantification of phosphorylated SMAD proteins in human blastocysts.","authors":"Todd Fallesen, A Sophie Brumm","doi":"10.1016/j.xpro.2025.103849","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103849","url":null,"abstract":"<p><p>The transforming growth factor β (TGF-β) signaling superfamily includes NODAL and bone morphogenetic protein (BMP) signaling, which lead to the phosphorylation of different SMAD proteins and regulate key developmental events. Here, we present a protocol for immunofluorescence detection of phosphorylated SMAD proteins combined with other transcription factors in pre-implantation human embryos. We describe steps for segmenting the nuclei in human blastocysts and quantifying their immunofluorescence intensity. This protocol can be adapted to investigate TGF-β superfamily signaling activity in other mammalian embryos or in vitro models of their development. For complete details on the use and execution of this protocol, please refer to Brumm et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103849"},"PeriodicalIF":1.3,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144182414","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
Protocol for detecting SnRK2 kinase activity in plants by immunoblotting, in-gel assay, band shift, and immunoprecipitation. 通过免疫印迹、凝胶内测定、条带移位和免疫沉淀检测植物中SnRK2激酶活性的方案。
IF 1.3
STAR Protocols Pub Date : 2025-05-28 DOI: 10.1016/j.xpro.2025.103842
Qingzhong Li, Xianping Yuan, Yang Zhao
{"title":"Protocol for detecting SnRK2 kinase activity in plants by immunoblotting, in-gel assay, band shift, and immunoprecipitation.","authors":"Qingzhong Li, Xianping Yuan, Yang Zhao","doi":"10.1016/j.xpro.2025.103842","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103842","url":null,"abstract":"<p><p>The SNF1-regulated protein kinase 2s (SnRK2s) are activated by phytohormone abscisic acid (ABA) and osmotic stress to control plant growth and stress responses; however, assessing SnRK2 activity is challenging. Here, we present a protocol to detect SnRK2 activity in plants. We describe steps for performing immunoblotting with anti-phospho-S175-SnRK2 antibody, in-gel kinase assay, band-shift assay, and immunoprecipitated kinase assay. Immunoblotting and in-gel kinase assays are suitable for evaluating endogenous SnRK2 activity, whereas band-shift and immunoprecipitated kinase assays are applicable to assess tagged SnRK2 activity in transgenic lines. For complete details on the use and execution of this protocol, please refer to Li et al.,<sup>1</sup> Li et al.,<sup>2</sup> and Yuan et al.<sup>3</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103842"},"PeriodicalIF":1.3,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144182474","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
Rapid and efficient protocol for optical clearing of mouse intestinal tissues for enhanced fluorescence imaging and 3D reconstruction. 快速和有效的方案光学清除小鼠肠道组织增强荧光成像和三维重建。
IF 1.3
STAR Protocols Pub Date : 2025-05-28 DOI: 10.1016/j.xpro.2025.103841
Joel Johnson George, Sierra H Ball, Elena Wang, Remy T Schneider, Claudia Capdevila, Francesca Martini, Hyeonjeong Lee, Jonathan Miller, Liang Cheng, John W Murray, Hans-Willem Snoeck, Kelley S Yan
{"title":"Rapid and efficient protocol for optical clearing of mouse intestinal tissues for enhanced fluorescence imaging and 3D reconstruction.","authors":"Joel Johnson George, Sierra H Ball, Elena Wang, Remy T Schneider, Claudia Capdevila, Francesca Martini, Hyeonjeong Lee, Jonathan Miller, Liang Cheng, John W Murray, Hans-Willem Snoeck, Kelley S Yan","doi":"10.1016/j.xpro.2025.103841","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103841","url":null,"abstract":"<p><p>Histological analysis of intestinal epithelial tissues is enhanced by 3D visualization compared to 2D sections. Here, we present a protocol for 3D visualization of intestinal epithelial cells using an optical clearing approach optimized for endogenous fluorescence and preservation of crypt-villus morphology. We describe steps for clearing and refractive index matching tissue. We provide detailed procedures for imaging and reconstructing tissue to visualize epithelial cells along the crypt-villus axis with high resolution. We illustrate this approach with endogenous tdTomato used for lineage tracing in the small intestine of Fgfbp1-CreER<sup>T2</sup>; Rosa26-tdTomato mice. For complete details on the use and execution of this protocol, please refer to Capdevila et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103841"},"PeriodicalIF":1.3,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144182127","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
Protocol to study ductal progenitor-like cells from the adult human pancreas using 3D suspension and methylcellulose-based culture systems. 使用3D悬浮液和甲基纤维素为基础的培养系统研究成人胰腺导管祖细胞样细胞的方案。
IF 1.3
STAR Protocols Pub Date : 2025-05-26 DOI: 10.1016/j.xpro.2025.103847
Heather N Zook, Janine C Quijano, Jose A Ortiz, Cecile Donohue, Neslihan Erdem, Hsun Teresa Ku
{"title":"Protocol to study ductal progenitor-like cells from the adult human pancreas using 3D suspension and methylcellulose-based culture systems.","authors":"Heather N Zook, Janine C Quijano, Jose A Ortiz, Cecile Donohue, Neslihan Erdem, Hsun Teresa Ku","doi":"10.1016/j.xpro.2025.103847","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103847","url":null,"abstract":"<p><p>Primary human ductal progenitor-like cells derived from donated pancreas have the potential to serve as a source of therapeutic insulin-producing beta cells for the treatment of diabetes. Here, we present a protocol for studying ductal progenitor-like cells using a good manufacturing practice (GMP)-compatible 3D suspension culture system and a methylcellulose- and Matrigel-based 3D colony assay. We describe steps for dispersing and cryopreserving human pancreatic cells and initiating and maintaining cultures. We then detail how to prepare ductal spheroids or colonies for downstream applications. For complete details on the use and execution of this protocol, please refer to Zook et al.<sup>1</sup> and Quijano et al.<sup>2</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103847"},"PeriodicalIF":1.3,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144162536","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
Protocol to perform multiplexed assays of variant effect using curated loci prime editing. 使用策划的基因座引物编辑进行多路变异效应测定的协议。
IF 1.3
STAR Protocols Pub Date : 2025-05-25 DOI: 10.1016/j.xpro.2025.103851
Carina G Biar, Nicholas Bodkin, Gemma L Carvill, Jeffrey D Calhoun
{"title":"Protocol to perform multiplexed assays of variant effect using curated loci prime editing.","authors":"Carina G Biar, Nicholas Bodkin, Gemma L Carvill, Jeffrey D Calhoun","doi":"10.1016/j.xpro.2025.103851","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103851","url":null,"abstract":"<p><p>Multiplexed assays of variant effect (MAVEs) perform simultaneous characterization of many variants. Here, we present a protocol to perform MAVEs using curated loci prime editing (cliPE), an accessible experimental pipeline that enables prime editing of a target gene. We describe steps for designing prime editing reagents, screening for genome editing efficiency, selecting a pool of cells edited to harbor different genetic variants, and sequencing. Lastly, we detail procedures for performing enrichment analysis to identify variants with normal or aberrant activity.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103851"},"PeriodicalIF":1.3,"publicationDate":"2025-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152134","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
Protocol for in vivo lineage tracing of regeneration-associated macrophages from injured skeletal muscle of adult mice. 成年小鼠损伤骨骼肌再生相关巨噬细胞的体内谱系追踪方法。
IF 1.3
STAR Protocols Pub Date : 2025-05-24 DOI: 10.1016/j.xpro.2025.103844
Neuza S Sousa, Pedro Sousa-Victor, Joana Neves
{"title":"Protocol for in vivo lineage tracing of regeneration-associated macrophages from injured skeletal muscle of adult mice.","authors":"Neuza S Sousa, Pedro Sousa-Victor, Joana Neves","doi":"10.1016/j.xpro.2025.103844","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103844","url":null,"abstract":"<p><p>Macrophages undergo phenotypic transitions that are essential for successful skeletal muscle (SkM) regeneration. Here, we present a protocol for in vivo lineage tracing of regeneration-associated macrophages, combining genetic labeling with transplantation of fluorescence-activated cell sorting (FACS)-isolated cells. Macrophages isolated from congenic CD45.1<sup>+</sup> donor mice are transplanted into pre-injured SkMs of CD45.2<sup>+</sup> mice and phenotyped by flow cytometry at designated time points of the regenerative process. We describe steps for muscle injury, SkM tissue processing, macrophage isolation, transplantation, and flow cytometry phenotyping. For complete details on the use and execution of this protocol, please refer to Sousa et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103844"},"PeriodicalIF":1.3,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144143794","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
Protocol for assessing the structural architecture, integrity, and cellular composition of murine bone ex vivo. 评估小鼠离体骨的结构结构、完整性和细胞组成的方案。
IF 1.3
STAR Protocols Pub Date : 2025-05-24 DOI: 10.1016/j.xpro.2025.103843
Jonathan W Lewis, Kathryn Frost, Georgiana Neag, Holly Adams, Emily Powell, Orla Gallagher, Ilaria Bellantuono, Amy J Naylor, Helen M McGettrick
{"title":"Protocol for assessing the structural architecture, integrity, and cellular composition of murine bone ex vivo.","authors":"Jonathan W Lewis, Kathryn Frost, Georgiana Neag, Holly Adams, Emily Powell, Orla Gallagher, Ilaria Bellantuono, Amy J Naylor, Helen M McGettrick","doi":"10.1016/j.xpro.2025.103843","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103843","url":null,"abstract":"<p><p>Understanding changes in murine bone is essential to comprehend bone homeostasis and the role of therapeutics in bone diseases. Here, we present a protocol to assess the structural architecture, integrity, and cellular composition of murine bone ex vivo. We describe steps for imaging and analyzing bone by micro-computed tomography (CT) and functionally testing bone strength through three-point bending. We then detail procedures for histology and analysis of calcein labeling. For complete details on the use and execution of this protocol, please refer to Lewis et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103843"},"PeriodicalIF":1.3,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144143792","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
Protocol for detecting peptide hormones in mosquito tissues. 蚊子组织中肽激素的检测方法。
IF 1.3
STAR Protocols Pub Date : 2025-05-24 DOI: 10.1016/j.xpro.2025.103850
Xiaoyi Dou, Kangkang Chen, Mark R Brown, Michael R Strand
{"title":"Protocol for detecting peptide hormones in mosquito tissues.","authors":"Xiaoyi Dou, Kangkang Chen, Mark R Brown, Michael R Strand","doi":"10.1016/j.xpro.2025.103850","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103850","url":null,"abstract":"<p><p>Peptide hormones in insects are primarily expressed in specialized brain, ventral nerve chord, and midgut cells. When released, peptide hormones play crucial roles in regulating physiology, reproduction, and behavior. Here, we present a protocol for detecting peptide hormones in mosquito tissues such as the brain, midgut, and hemolymph. We describe steps for tissue preparation, immunocytochemistry, fluorescent quantification, and enzyme-linked immunoassay (EIA). This protocol offers a versatile and effective approach for studying peptide hormone expression and abundance in insects. For complete details on the use and execution of this protocol, please refer to Dou et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103850"},"PeriodicalIF":1.3,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144143793","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
Protocol for RNA extraction from glioblastoma cell-line-derived spheroids embedded in atelocollagen gel guided by real-time imaging. 实时成像引导下从胶质母细胞瘤细胞系衍生球体中提取RNA的方案。
IF 1.3
STAR Protocols Pub Date : 2025-05-24 DOI: 10.1016/j.xpro.2025.103839
Mayumi Fujita, Kaori Imadome, Tetsuro Sato, Hirokazu Hirakawa, Junko Kado, Asako Yamagiri, Taichi Miura, Satoshi Kamimura
{"title":"Protocol for RNA extraction from glioblastoma cell-line-derived spheroids embedded in atelocollagen gel guided by real-time imaging.","authors":"Mayumi Fujita, Kaori Imadome, Tetsuro Sato, Hirokazu Hirakawa, Junko Kado, Asako Yamagiri, Taichi Miura, Satoshi Kamimura","doi":"10.1016/j.xpro.2025.103839","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103839","url":null,"abstract":"<p><p>Microscopy advancements enable real-time imaging and analysis of cancer spheroids embedded in atelocollagen gel. Here, we present a protocol for extracting high-quality total RNA from 3D tumor spheroids embedded in atelocollagen gel, including spheroids and invading cells dispersed into surrounding gel. We describe steps for spheroid formation, gel embedding, live-cell imaging, and image analysis. We then detail procedures for collecting sufficient quantities of total RNA suitable for comprehensive transcriptome analysis. This approach advances transcriptome profiling in diverse 3D cultured samples.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103839"},"PeriodicalIF":1.3,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144143795","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
Protocol to investigate sensorimotor features in young children with autism using functional and resting-state fMRI data from the NIMH Data Archive. 使用来自NIMH数据档案的功能和静息状态fMRI数据研究幼儿自闭症感觉运动特征的方案。
IF 1.3
STAR Protocols Pub Date : 2025-05-23 DOI: 10.1016/j.xpro.2025.103827
Kristina Denisova, Daniel M Wolpert
{"title":"Protocol to investigate sensorimotor features in young children with autism using functional and resting-state fMRI data from the NIMH Data Archive.","authors":"Kristina Denisova, Daniel M Wolpert","doi":"10.1016/j.xpro.2025.103827","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103827","url":null,"abstract":"<p><p>Head movements cause artifacts in infant neuroimaging that can often render acquired data unusable. Here, we present a protocol that harnesses head motion from fMRI and resting-state (rs)-fMRI NIMH Data Archive (NDA) data to obtain quantitative measures of sensorimotor function in young children and associate them with future cognitive and autism outcomes. We describe steps for downloading, organizing, and pre-processing fMRI data to yield data on in-scan head motion. We then detail procedures for preparing phenotypic data to link with sensorimotor data. For complete details on the use and execution of this protocol, please refer to Denisova and Wolpert.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103827"},"PeriodicalIF":1.3,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144143796","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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