Bio-protocol最新文献

{"title":"Workflow for a Functional Assay of Candidate Effectors From Phytopathogens Using a TMV-GFP-based System.","authors":"Peng Cao, Haotian Shi, Jialan Chen, Langjun Cui, Meixiang Zhang, Yuyan An","doi":"10.21769/BioProtoc.5287","DOIUrl":"https://doi.org/10.21769/BioProtoc.5287","url":null,"abstract":"<p><p>The ability to efficiently screen plant pathogen effectors is crucial for understanding plant-pathogen interactions and developing disease-resistant crops. Traditional methods are often labor-intensive and time-consuming. Here, we present a robust, high-throughput screening assay using the tobacco mosaic virus-green fluorescent protein (TMV-GFP) vector system. The screening system combines the TMV-GFP vector and <i>Agrobacterium</i>-mediated transient expression in the model plant <i>Nicotiana benthamiana</i>. This system enables the rapid identification of effectors that interfere with plant immunity (both activation and suppression). The biological function of these effectors can be easily evaluated within six days by observing the GFP fluorescence signal using a UV lamp. This protocol significantly reduces the time required for screening and increases the throughput, making it suitable for large-scale studies. The method is versatile, cost-effective, and can be adapted to effectors with immune interference activity from various pathogens. Key features • A robust, cost-effective, and high-throughput functional screening system for plant pathogen effectors. • Utilizes the TMV-GFP vector for rapid monitoring of effector activity. • Evaluates the function of effectors within a few days using just a UV lamp. • Adaptable to both apoplastic and cytoplasmic effectors from various phytopathogens.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5287"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021684/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144002327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER).","authors":"Hirotaka Yamamoto, Tomokazu Tamura, Takausuke Fukuhara","doi":"10.21769/BioProtoc.5275","DOIUrl":"https://doi.org/10.21769/BioProtoc.5275","url":null,"abstract":"<p><p>Reverse genetics systems in virology are technologies used to generate recombinant viruses, enabling the manipulation of viral genes. Recombinant viruses facilitate the investigation of pathogenesis and the development of antivirals. In studies of positive-sense single-stranded RNA (ssRNA) viruses, a reverse genetics approach typically uses infectious viral cDNA clones derived from bacterial artificial chromosomes and plasmids or from the in vitro ligation of viral cDNA fragments. However, these methods are time-consuming, involve complex procedures, and do not always successfully generate recombinant viruses. Possible reasons for unsuccessful outcomes include i) viral sequences exhibiting toxicity in bacterial systems, ii) the duplication of viral genes observed in some strains, complicating the acquisition of correct cDNA clones, and iii) certain cell lines being highly susceptible to infection but difficult to transfect with nucleotides. For these reasons, a simple and rapid reverse genetics system is needed to accelerate research on ssRNA viruses. The circular polymerase extension reaction (CPER) method offers a solution by eliminating the need for molecular cloning in bacteria, enabling the generation of recombinant viruses over a shorter timeframe. This method has been widely adopted for the study of ssRNA viruses, including SARS-CoV-2 and flaviviruses. Recently, we expanded the CPER method for ssRNA viruses using internal ribosome entry site (IRES)-mediated translation. This protocol details the experimental procedures, using bovine viral diarrhea virus as an example-one of the most challenging viruses for generating viral cDNA clones because of the factors listed above. Key features • Rapid generation of recombinant positive-strand RNA viruses. • The CPER method eliminates the need for molecular cloning in bacteria, enabling the rapid generation of recombinant viruses. • The CPER method for ssRNA viruses enables efficient translation of viruses using IRES by incorporating the gene cassette of RNA Pol-I promoters and terminators.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5275"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021584/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144000515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"X-Ray Photon Correlation Spectroscopy, Microscopy, and Fluorescence Recovery After Photobleaching to Study Phase Separation and Liquid-to-Solid Transition of Prion Protein Condensates.","authors":"Mariana J do Amaral, Aline R Passos, Satabdee Mohapatra, Maria Heloisa Freire, Susanne Wegmann, Yraima Cordeiro","doi":"10.21769/BioProtoc.5277","DOIUrl":"https://doi.org/10.21769/BioProtoc.5277","url":null,"abstract":"<p><p>Biomolecular condensates are macromolecular assemblies constituted of proteins that possess intrinsically disordered regions and RNA-binding ability together with nucleic acids. These compartments formed via liquid-liquid phase separation (LLPS) provide spatiotemporal control of crucial cellular processes such as RNA metabolism. The liquid-like state is dynamic and reversible, containing highly diffusible molecules, whereas gel, glass, and solid phases might not be reversible due to the strong intermolecular crosslinks. Neurodegeneration-associated proteins such as the prion protein (PrP) and Tau form liquid-like condensates that transition to gel- or solid-like structures upon genetic mutations and/or persistent cellular stress. Mounting evidence suggests that progression to a less dynamic state underlies the formation of neurotoxic aggregates. Understanding the dynamics of proteins and biomolecules in condensates by measuring their movement in different timescales is indispensable to characterize their material state and assess the kinetics of LLPS. Herein, we describe protein expression in <i>E. coli</i> and purification of full-length mouse recombinant PrP, our in vitro experimental system. Then, we describe a systematic method to analyze the dynamics of protein condensates by X-ray photon correlation spectroscopy (XPCS). We also present fluorescence recovery after photobleaching (FRAP)-optimized protocols to characterize condensates, including in cells. Next, we detail strategies for using fluorescence microscopy to give insights into the folding state of proteins in condensates. Phase-separated systems display non-equilibrium behavior with length scales ranging from nanometers to microns and timescales from microseconds to minutes. XPCS experiments provide unique insights into biomolecular dynamics and condensate fluidity. Using the combination of the three strategies detailed herein enables robust characterization of the biophysical properties and the nature of protein phase-separated states. Key features • For FRAP in cells, we recommend using a spinning disk confocal microscope coupled with temperature and CO₂ incubator. • For fluorescence microscopy, we recommend simultaneously imaging differential interference contrast (DIC) (or phase contrast) and fluorescence channels to obtain morphological details of phase-separated structures. • For XPCS, coherent X-ray beams, fast X-ray detectors in fourth and third synchrotron light sources, and X-ray free-electron lasers are required.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5277"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021588/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144053831","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Robust and Easy Protein Purification Method Using SpyDock-Modified Resin.","authors":"Xiaofeng Yang, Zhanglin Lin, Ya Xiang, Binrui Chen, Zisha Lao","doi":"10.21769/BioProtoc.5270","DOIUrl":"https://doi.org/10.21769/BioProtoc.5270","url":null,"abstract":"<p><p>Protein purification is a critical step in both life sciences and biomanufacturing. Traditional affinity chromatography (AC) methods, including His-tag-based purification, provide high-purity proteins but are limited by the high cost of resins and the need for additional tag-removal steps. In this protocol, we present a reusable SpyDock-modified epoxy resin coupled with a pH-inducible self-cleaving intein for direct purification of proteins with authentic N-termini. This method enables efficient protein purification from cell lysates, achieving high purity (>90%) and yields comparable to the His-tag approach, without requiring tag removal. The SpyDock-modified resin protocol is robust, easy to implement, and cost-effective, making it suitable for both research and large-scale industrial applications. Key features • This protocol offers a robust and straightforward method for purifying proteins with authentic N-termini, eliminating the need for additional tag removal steps. • The approach achieves higher purity and comparable yields to the commercial His-tag method. • The SpyDock-modified epoxy resin is easy to prepare, cost-effective, and reusable.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5270"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021586/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144065363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Scalable Alkaline Extraction Protocol for Microbial DNA Screening by PCR.","authors":"Stavroula Goudoudaki, Manousos E Kambouris, Stavroula Kritikou, Afroditi Milioni, Aristea Velegraki, Yiannis Manoussopoulos, George P Patrinos","doi":"10.21769/BioProtoc.5290","DOIUrl":"https://doi.org/10.21769/BioProtoc.5290","url":null,"abstract":"<p><p>In molecular diagnosis, DNA extraction kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Previous fast extraction protocols such as detergent-based ones allow fast DNA extraction for nucleic acid amplification tests (NAAT), mainly polymerase chain reaction (PCR). The use of NaOH (dense alkali) to rupture cells and nuclei and destabilize the conformation of DNases might alleviate shortages and costs while retaining enough robustness to treat complicated samples with minimal environmental and logistical footprint. Biological samples are hand-crushed using a pestle in 1.5 mL tubes with 360 μL of 0.2 M NaOH for 3-5 min and incubated at 75 °C for 10 min. For immediate use, 115.2 μL of 1 M Tris (pH 8) and 364.8 μL nuclease-free water are added, and the sample is vortexed for 10 s and spun at 10,000× <i>g</i> for 3 min; then, 700 μL is transferred to a clean microtube. Two serial dilutions follow, and all concentrations are used as templates for PCR. A refined, storable extract can be produced by adding 70 μL of HCl 1 M (instead of Tris-HCl) and one volume of cold isopropanol to the extract for standard precipitation. This method can increase throughput in emergencies by field deployment in resource-limited settings (RLS) or allow benchtop backup in cases of acquisition disruption or sample surge in established facilities. The crude extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing NAAT in resource-limited settings with low costs and waste footprint or during prolonged crises, where supply chain failures may occur. The refined version produces alcohol-precipitated nucleic acids, suitable for both immediate use and for storage or dispatch for spatiotemporally separate analysis while offering much better amplification quality with a small increase in time and minimal increase in expendables/chemicals needed. Key features • DNA extraction from different sample types using only boiling water and occasional mechanical assistance. • Crude extract serially diluted to bypass purification and quantification steps. • Refined extract is partly purified, more enriched, storable, and transportable and contributes to higher sensitivity. • Both versions decrease costs and the overall footprint of testing to increase sustainability in field operations and in standard lab environments under supply chain derailment.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5290"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021680/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144047189","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Vitro Bone Marrow-Derived Dendritic Cells (BMDC) Generation for Antigen Presentation Assay.","authors":"Sudhakar Singh, Azeez Tehseen, Mohammed Shaaz Iqbal, Sharvan Sehrawat","doi":"10.21769/BioProtoc.5278","DOIUrl":"https://doi.org/10.21769/BioProtoc.5278","url":null,"abstract":"<p><p>Dendritic cells (DC) are sentinel cells of the immune system that process and present antigens to activate T cells, thus serving to bridge the innate and adaptive immune systems. DCs are particularly efficient at cross-presentation whereby exogenously acquired antigens are processed and presented in context with MHCI molecules to activate CD8⁺ T cells. Assaying antigen presentation by DCs is a critical parameter in assessing immune functionality. However, the low abundance of bona fide DCs within the lymphoid compartments limits the utility of such assays. An alternative approach employing the culturing of bone marrow cells in the presence of factors needed for DC lineage commitment can result in the differentiation of bone marrow dendritic cells (BMDCs). This protocol details the process of in vitro generation of BMDCs and demonstrates their subsequent utility in antigen presentation assays. The protocol described can be adapted to various conditions and antigens. Key features • BMDCs can serve as surrogate antigen-presenting cells (APCs) for assessing in vitro and in vivo antigen presentation. • Co-culture of antigen-stimulated BMDCs with CFSE-labeled T cells can help quantify the responsiveness of both the antigen presenters and responders. • In vivo analysis of antigen presentation by BMDCs can be assessed using an adoptive transfer approach. • CFSE labeling can help track in vivo the fate of adoptively transferred BMDCs as well as T cells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5278"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021583/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144047999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Endo-1,4-β-D-xylanase Assay Using Azo-Xylan and Variants Thereof.","authors":"Luca Bombardi, Annalaura Coltro, Salvatore Fusco","doi":"10.21769/BioProtoc.5283","DOIUrl":"https://doi.org/10.21769/BioProtoc.5283","url":null,"abstract":"<p><p>Xylan is the main component of hemicellulose and consists of a complex heteropolysaccharide with a heterogeneous structure. This framework, in addition to the crystalline structure of cellulosic fibers and the rigidity of lignin, makes lignocellulosic biomass (LCB) highly recalcitrant to degradation. Xylanases are glycoside hydrolases that cleave the β-1,4-glycoside linkages in the xylan backbone and have attracted increasing attention due to their potential uses in various industrial sectors such as pulp and paper, baking, pharmaceuticals, and lignocellulosic biorefining. For decades, the measurement of xylanase activity was based on reducing sugar quantification methods like DNS or Nelson/Somogyi assays, with numerous limitations in terms of specificity and interference from other enzymatic activities. A better alternative is the colorimetric Azo-Xylan assay, which specifically measures the endo-1,4-β-D-xylanase activity. In this study, the Azo-Xylan protocol was adapted from the company Megazyme to determine the enzymatic activity of thermostable xylanases produced by microbial consortia (i.e., microbiomes), aiming to determine biochemical features such as temperature and pH optima, thermostability, and shelf life. This modified approach offers a rapid, cost-effective, and highly specific method for the determination of xylanase activity in complex mixtures, helping the development of a xylanase-based method for the hydrolysis of hard-degrading substrates in bio-based industries. Key features • Direct enzyme assay for qualitative xylanase activity detection or quantitative measurement with a calibration curve. • Specific for determination of endo-1,4-β-D-xylanase activity, allowing to overcome interferences by enzymes with other activities.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5283"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021678/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144060923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and Culture of Primary Pericytes from Mouse.","authors":"Tamara McErlain, Cristina M Branco, Meera Murgai","doi":"10.21769/BioProtoc.5288","DOIUrl":"https://doi.org/10.21769/BioProtoc.5288","url":null,"abstract":"<p><p>Pericytes are essential for tissue homeostasis, functioning to regulate capillary blood flow. Dysfunctional pericytes are implicated in various pathologies, including cancer progression. Despite their important function in both health and disease, pericytes remain understudied due to a lack of robust model systems that accurately reflect their in vivo biology. Here, we present a comprehensive protocol for isolating and culturing primary pericytes from murine lung, brain, bone, and liver tissues, based on NG2 expression using an antibody-conjugated magnetic bead approach. Our protocol emphasizes the importance of physiological oxygen tension during ex vivo culture (10% O₂ for lung pericytes and 5% O₂ for brain, bone, and liver pericytes). These conditions stabilize the expression of characteristic pericyte markers at both the transcriptional and protein levels. Importantly, we optimized growth conditions to limit the expression of the plasticity factor <i>Klf4</i> in order to prevent spontaneous phenotypic switching in vitro. This protocol provides a reliable and reproducible method for obtaining pericytes suitable for high-throughput analyses in order to explore pericyte biology in both physiological and pathological contexts. Key features • Isolation of primary pericytes from mouse lung, brain, bone, and liver. • Emphasis on physioxic culturing conditions to better maintain pericyte phenotype. • Representative of pericyte biology in both health and disease contexts.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5288"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021682/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144065390","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Novel Optimized Silver Nitrate Staining Method for Visualizing and Quantifying the Osteocyte Lacuno-Canalicular System (LCS).","authors":"Jinlian Wu, Chunchun Xue, Qiang Li, Hongjin Wu, Jie Zhang, Chenglong Wang, Weiwei Dai, Libo Wang","doi":"10.21769/BioProtoc.5289","DOIUrl":"https://doi.org/10.21769/BioProtoc.5289","url":null,"abstract":"<p><p>The osteocyte lacuno-canalicular system (LCS) plays a crucial role in maintaining bone homeostasis and mediating cellular mechanotransduction. Current histological techniques, particularly the Ploton silver nitrate staining method, face challenges such as variations in solution concentrations and types as well as a lack of standardization, which limits their broader application in osteocyte research. In this study, we present a simplified and more effective silver nitrate staining protocol designed to address these issues. Our method utilizes a 1 mol/L silver nitrate solution combined with optimized gelatin-formic acid solutions at varying concentrations (0.05%-0.5% type-B gelatin and 0.05%-5% formic acid, or 1%-2% type-B gelatin and 0.1%-2% formic acid). Staining is performed for 1 h under 254 nm ultraviolet light or 90 min under room light, followed by washing with Milli-Q water to terminate staining. This novel optimized method yields consistent and distinct staining of the osteocyte LCS across multiple species, demonstrating superior efficiency and reliability compared to the Ploton method. It will significantly advance research in osteocyte biology and provide a valuable tool for exploring the adaptive evolution of osteocyte LCS morphology and function across various taxa. Key features • A novel optimized silver nitrate method using a 1 mol/L silver nitrate solution with type-B gelatin-formic acid solution effectively stains the osteocyte LCS. • The novel optimized method is more efficient for staining the osteocyte LCS across different species. • The novel optimized method is simpler to perform and more cost-effective than conventional methods. Graphical overview <b>Overview of the silver nitrate staining method for staining and qualifying osteocyte lacuno-canalicular system (LCS).</b> Created in BioRender. Libo, W. (2024) https://BioRender.com/c10z002.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5289"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021683/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143994276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis and Quantification of Functional Regeneration of Dendrite and Axon of PVD Neuron After Laser Injury in <i>Caenorhabditis elegans</i>.","authors":"Harjot Kaur Brar, Dikshalee Bassi, Anindya Ghosh-Roy","doi":"10.21769/BioProtoc.5280","DOIUrl":"https://doi.org/10.21769/BioProtoc.5280","url":null,"abstract":"<p><p>Research into nervous system injuries and regeneration has emerged as a crucial field of study. In many cases such as trauma or stroke, both axons and dendrites are equally damaged; however, studying injury and repair mechanisms in both neurite processes (axons and dendrites) of the same neuron has been challenging. Additionally, correlating the behavioral aspects of neuronal injury with anatomical regeneration is important for a better understanding of the functional rewiring process. Here, we describe protocols for injuring the dendrites and the axon of the PVD neuron of <i>C. elegans</i> using a two-photon infrared (IR) femtosecond laser system, and subsequent imaging of injured neurites during the course of regeneration. Additionally, we describe the protocols for the behavioral study concerning the PVD neuron and their analysis, which can offer valuable insights. These assays can be implemented to assess the function of the pathways that play specific roles in dendrite vs. axon regeneration. Key features • Dendritic and axonal injury in PVD neurons using a two-photon microscope. • Behavioral analysis involving harsh touch response and proprioception following neurite injury. • Correlation of dendrite and axon regeneration events with behavioral parameters.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5280"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021587/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144000468","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}