Bio-protocol最新文献

{"title":"Efficient and Site-Specific Incorporation of 3-Nitro-Tyrosine Into Recombinant Proteins in <i>Escherichia coli</i>.","authors":"Sarah B McGee, Stanislau Stanisheuski, Ryan A Mehl, Richard B Cooley","doi":"10.21769/BioProtoc.5674","DOIUrl":"10.21769/BioProtoc.5674","url":null,"abstract":"<p><p>3-nitro-tyrosine (nitroTyr) is one of numerous oxidative protein modifications implicated in diseases such as cardiovascular disease, cancer, and amyotrophic lateral sclerosis (ALS). Because of this, the ability to site-specifically encode nitroTyr into recombinant proteins is a powerful approach for studying these disease pathways. However, producing proteins with defined nitration sites is technically challenging due to the limitations of traditional chemical nitration via peroxynitrite, which lacks residue and site-specificity. Genetic code expansion (GCE) offers a solution by enabling precise incorporation of nitroTyr at designated TAG codons using engineered aminoacyl-tRNA synthetase/tRNA pairs from <i>Methanocaldococcus jannaschii</i> and <i>Methanomethylophilus alvus</i>. This protocol provides a reliable, optimized workflow for incorporating nitroTyr into proteins in <i>E. coli</i> using GCE. It guides users through key considerations in selecting cell lines, media conditions, and GCE systems to minimize off-target effects such as release factor 1 competition, near-cognate suppression, and chemical reduction of nitroTyr. The method is demonstrated using wild-type and TAG-containing superfolder GFP but is broadly applicable to other proteins of interest. Key features • This protocol offers a practical guide for the recombinant expression of proteins containing site-specific 3-nitro-tyrosine in <i>E. coli.</i> • These methods should be used to characterize the functional and structural consequences of site-specific tyrosine nitration on proteins without having to modify any other residues. • This protocol avoids the use of peroxynitrite as a method to nitrate proteins, which modifies all solvent accessible tyrosine residues to different extents. • Users are guided through the advantages and disadvantages of using different expression strains and genetic code expansion systems depending on specific needs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5674"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103966/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792110","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":"TIE-UP-SIN: A Method for Enhanced Identification of Protein-Protein Interactions.","authors":"Maximilian Schedlowski, Stephan Michalik, Tilly Hoffmüller, Marco Harms, Leif Steil, Kristin Surmann, Christian Hentschker, Manuela Gesell Salazar, Uwe Völker, Alexander Reder","doi":"10.21769/BioProtoc.5663","DOIUrl":"https://doi.org/10.21769/BioProtoc.5663","url":null,"abstract":"<p><p>Protein-protein interactions (PPIs) govern nearly all aspects of cellular physiology, yet identifying these interactions under native conditions remains challenging. Here, we present TIE-UP-SIN (targeted interactome experiment for unknown proteins by stable isotope normalization), a robust method for in vivo identification and quantification of PPIs in bacterial systems. The protocol combines metabolic labeling with ¹⁵N isotopes, reversible formaldehyde crosslinking, affinity purification, and quantitative mass spectrometry. TIE-UP-SIN preserves transient or weak interactions during purification and quantifies interaction partners using internal light/heavy peptide ratios, reducing experimental variability. The method employs a triple-sample design to distinguish specific from nonspecific interactors and can be adapted to various bacterial species and affinity tags. Data analysis is streamlined through a user-friendly web application (https://shiny-fungene.biologie.uni-greifswald.de/TIE_UP_SIN_app) that automates statistical analysis, normalization, and visualization, requiring no programming expertise. The entire workflow from cell culture to mass spectrometry data acquisition takes approximately 4-5 days, with data analysis completed in 1-2 days using the web application. Key features • Captures transient protein interactions in vivo through reversible formaldehyde crosslinking under native expression conditions. • Internal ¹⁵N metabolic labeling enables robust quantification and reduces experimental variability across biological replicates. • Triple-sample design (WT/WT, bait/WT, bait/bait) distinguishes specific from nonspecific interactors with high confidence. • Applicable to diverse bacterial systems with simple adaptation to any affinity-tagged bait protein.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5663"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103894/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792121","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":"Preparation and Assembly of the Axial Invasion Chamber for Live-Cell Invadopodia Imaging.","authors":"Mark Garewal, Kenneth A Myers","doi":"10.21769/BioProtoc.5658","DOIUrl":"https://doi.org/10.21769/BioProtoc.5658","url":null,"abstract":"<p><p>Metastasis is initiated by cell invasion of the basement membrane, facilitating cell migration and colonization at a secondary tumor site. Cancer cells remodel the cytoskeleton to form ventral protrusions, termed invadopodia, that traffic and deliver matrix metalloproteases to degrade the extracellular matrix. Traditional efforts have utilized immunolabeling to measure protein localization within invadopodia, an approach limited by reduced temporal resolution, logistical challenges in orienting invadopodia within the focal plane of the objective lens, and impaired ability to reconstitute physiological conditions. Here, we describe a protocol for constructing and utilizing the axial invasion chamber (AIC) to perform live-cell 3D visualization of mature elongating invadopodia under physiological conditions. The AIC is simple to build, using standard 35 mm glass-bottom dishes that suit most microscope stage holders. A polyester membrane is used to uniformly orient and promote invadopodia formation and restrict cell migration. The AIC extracellular matrix is composed of readily available reagents that have been optimized to facilitate cell adhesion and invadopodia maturation. Critical advances of the AIC include imaging and measurements of protein localization without immunolabeling, imaging of live cell invadopodia using conventional inverted microscopes, and production of a fully operational apparatus within 28 h from initial assembly. While the protocol has been used for live-cell invadopodia protein localization and structure, it provides an opportunity to interchange components of the polyester membrane and/or the extracellular matrix to optimize the device for a variety of different cell types and cell invasion studies. Key features • Enables high-resolution live-cell invadopodia imaging along the axial plane and visualization of protein localization and length of protrusion. • Live-cell imaging with transient transfection of fluorescent proteins and interchangeable components to study various aspects of cell invasion and migration.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5658"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103895/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792139","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":"Spatial Imaging and Quantification of Hydrogen Peroxide in <i>Arabidopsis</i> Roots: From Sample Preparation to Image Analysis.","authors":"Mario Fenech, Vitor Amorim-Silva","doi":"10.21769/BioProtoc.5659","DOIUrl":"https://doi.org/10.21769/BioProtoc.5659","url":null,"abstract":"<p><p>Reactive oxygen species (ROS) are central regulators of plant development and stress responses, with hydrogen peroxide (H₂O₂) acting as a key signaling molecule whose spatial distribution determines adaptive versus damaging outcomes. Accurate detection of H₂O₂ at tissue and cellular resolution is therefore essential for understanding redox-dependent regulation of plant growth. A variety of techniques have been used to monitor H₂O₂, including bulk spectrophotometric and fluorometric assays, genetically encoded sensors for real-time measurements, and chemical probes for in situ detection. While these approaches differ in sensitivity, specificity, and temporal resolution, many are limited by a lack of spatial information, technical complexity, or dependence on transgenic material. Here, we present a detailed protocol for 3,3'-diaminobenzidine (DAB)-based histochemical detection of H₂O₂ in seedling roots, covering staining, imaging, and semi-quantitative image analysis using open-source software (FIJI/ImageJ). The method relies on peroxidase-mediated oxidation of DAB, resulting in a stable, light-resistant, and insoluble precipitate that enables visualization of H₂O₂ accumulation with high spatial resolution. This protocol provides a robust, accessible, and genetically independent approach for spatial analysis of H₂O₂ in plant tissues. Its simplicity, compatibility with diverse genotypes and treatments, and suitability for semi-quantitative analysis make it a valuable tool for examining the spatial distribution of H₂O₂, thereby providing spatial insight into redox-related regulatory processes during plant development and stress responses. Key features • Built upon methods developed by Thordal-Christensen et al. [1] and Daudi and O'Brien [2], with a specific focus on root staining. • Includes a downstream image analysis pipeline for semi-quantitative H₂O₂ measurement in DAB-stained roots using the open-source software FIJI/ImageJ. • Provides detailed, step-by-step video tutorials for image analysis in FIJI/ImageJ. • Includes a Fiji/ImageJ script (Macro 1) for automating the application of fixed-intensity scaling using Spectrum LUT.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5659"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103900/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792081","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 Simple and Easy Method for RNA Extraction from the Cyanobacterium <i>Synechocystis</i> sp. PCC 6803.","authors":"Bharat Kumar Majhi, Julian J Eaton-Rye","doi":"10.21769/BioProtoc.5654","DOIUrl":"https://doi.org/10.21769/BioProtoc.5654","url":null,"abstract":"<p><p>Cyanobacteria have been widely used as model organisms in photobiochemical research and have recently been exploited as hosts in numerous pilot studies to produce valuable biochemicals via genetic and metabolic modifications. Analyzing cellular RNA is a suitable method for studying genetic changes in cells. Several methods have previously been reported for cyanobacterial RNA extraction. However, the majority of these methods rely heavily on phenol and chloroform, which are hazardous. Additionally, these methods are time-consuming and difficult to perform. Using <i>Synechocystis</i> sp. PCC 6803 as a model, this study developed a novel method for extracting total ribonucleic acid (RNA) using standard centrifugation techniques and laboratory chemicals such as citric acid, ethylenediaminetetraacetic acid, sodium dodecyl sulfate, sodium chloride, and tri-sodium citrate dihydrate to extract RNA from cyanobacterial cells. This method does not necessitate the use of hazardous chemicals, especially phenol and chloroform. Furthermore, it is cost-effective since it does not require expensive chemicals. The results of the quantification, purity, and integrity checks show the effectiveness of this method for extracting good-quality RNA. Furthermore, RT-qPCR results demonstrate that the quality of the extracted RNA is suitable for downstream applications. Key features • Simple and efficient RNA extraction method. • Requires less than an hour to extract total RNA. • Provides high-quality RNA suitable for downstream applications. • This method might be used to extract RNA from other cyanobacteria and algae.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5654"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067156/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679391","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 Male Mouse Model of WIN 55,212-2 Self-Administration to Study Cannabinoid Addiction.","authors":"Elena Martín-García, María Fernanda Ponce-Beti, Tatiana Gusinskaia, Alba López-Moraga, Roberto Capellán, Rafael Maldonado","doi":"10.21769/BioProtoc.5652","DOIUrl":"https://doi.org/10.21769/BioProtoc.5652","url":null,"abstract":"<p><p>Despite substantial progress in preclinical cannabinoid research, translational studies on cannabis use disorders (CUD) are still insufficient due to the absence of robust, validated animal models that fully recapitulate the multifactorial clinical phenotype of human CUD. The complex nature of CUD and the incomplete understanding of its underlying neurobiological mechanisms contribute to the limited availability of effective treatments. To address this gap, we developed an operant conditioning-based mouse model that enables the identification of individual vulnerability or resilience to CUD development. This highly translational model is based on the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) criteria for substance use disorders. The model allows the assessment of addiction-like behaviors by evaluating three behavioral domains: 1) persistence of responding during periods of cannabinoid unavailability, 2) motivation for cannabinoid seeking measured using a progressive ratio schedule, and 3) compulsivity, assessed when cannabinoid reward is paired with an aversive consequence such as a mild electric foot shock. A major strength of this paradigm is its ability to quantify two phenotypic traits proposed as predisposing factors for addiction vulnerability and two parameters related to craving. In addition, the model is specifically designed to evaluate genetic and circuit-level manipulations using chemogenetic approaches, with minor modifications required by surgical viral-vector delivery. Using this protocol, we can determine whether altering the excitability of specific neural networks promotes resilience or vulnerability to developing cannabinoid addiction. Elucidating these mechanisms is expected to facilitate the identification of novel and more effective therapeutic interventions for CUD. Key features • Operant conditioning-based mouse model to study cannabis use disorders (CUD) based on DSM-5 substance use disorder criteria. • Enables assessment of addiction-like behaviors across persistence, motivation (progressive ratio), and compulsivity under punishment, allowing stratification of vulnerable versus resilient individuals. • Quantifies phenotypic traits linked to cannabinoid addiction vulnerability and behavioral signatures associated with craving for cannabinoids. • Compatible with genetic and circuit-level manipulations to test how specific neural networks modulate CUD-related behaviors.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5652"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067159/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679382","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, Culture, and Differentiation of Bovine Muscle Resident Stem Cells.","authors":"Perri Gish, Madison W Stewart, Maykal Tsonov, Brandon Khuu, Rachel Espinoza, Payam Vahmani, Lucas R Smith","doi":"10.21769/BioProtoc.5647","DOIUrl":"https://doi.org/10.21769/BioProtoc.5647","url":null,"abstract":"<p><p>Bovine muscle satellite cells (MuSC) and fibro-adipogenic progenitor cells (FAP) are muscle resident stem cells that are responsible for postnatal muscle growth, intramuscular fat deposition, and extracellular matrix generation. These cells are of increasing interest for the cultivated meat community due to their ability to generate all the major components of meat; additionally, these cells are of interest to conventional animal science research to elucidate mechanisms to improve meat quality. To use these cells for these goals, efficient and accurate cell isolation, culture, and differentiation are essential to evaluate their cell fate decisions and behaviors. In this protocol, we detail a simultaneous isolation of both MuSCs and FAPs with multiple intermediate stopping points, allowing for flexibility for day-of time constraints. We also detail improved growth conditions to maximize cell expansion and procedures to assess cell differentiation. This protocol provides a flexible isolation procedure that is compatible with sampling in modern slaughterhouses or from biopsies. Additionally, the differentiation procedures provide improved differentiation but still allow in vitro treatment and assessment. Key features • This protocol offers a flexible in-lab procedure to isolate bovine FAPs and MuSCs from tissue collected post-slaughter with multiple pause points. • The protocol demonstrates successful conditions to grow, expand, and differentiate bovine FAPs with an optimized adipogenic differentiation medium. • Strategies for planning your primary cell isolation, choosing the sampling location, and characterizing differentiation of bovine FAPs and MuSCs are included.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5647"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13070837/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147693732","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":"Denaturing SUMO Immunoprecipitation From Mitotic Cells.","authors":"Alexandra K Walker, Alexander J Lanz, Joanna R Morris","doi":"10.21769/BioProtoc.5648","DOIUrl":"https://doi.org/10.21769/BioProtoc.5648","url":null,"abstract":"<p><p>Small ubiquitin-related modifiers (SUMOs) are covalently conjugated onto the proteome and serve as signaling molecules in many aspects of eukaryotic cell biology, from <i>S. cerevisiae</i> and <i>C. elegans</i> to <i>H. sapiens</i>. The conjugatable SUMO variants, SUMO1 and the almost identical SUMO2 and SUMO3 (designated SUMO2/3), are processed by an E1(SAE1:SAE2)-E2(UBC9)-E3 enzyme cascade to produce SUMO-modified proteins. The prerogative of the SUMO biology field is to identify and study the specific proteins undergoing SUMOylation, which grants us insights into the biological pathway of interest. This protocol was developed using the human osteosarcoma cell line U2OS to enable the investigation of SUMO conjugates in mitosis, the cell division phase of the cell cycle. We enrich the cell population for mitotic cells, which are isolated and subjected to stringent lysis conditions involving a high concentration of SDS and DTT in RIPA buffer, to promote complete protein denaturation. The lysates in high SDS RIPA buffer are diluted to reduce the overall SDS concentration and undergo conventional immunoprecipitation using SUMO1- or SUMO2/3-specific antibodies bound to protein A/G agarose beads. The samples are then compatible with downstream readouts such as western blots and mass spectrometry. This protocol detects endogenous SUMOylated proteins and avoids exogenous SUMO overexpression, which can alter SUMO conjugate formation. Furthermore, this denaturing protocol ensures only SUMOylated proteins are immunoprecipitated, and not their interactors. Key features • Purifies endogenous SUMO-modified proteins by building on Becker et al. [1]. • Enriches and isolates cells in mitosis using nocodazole and mitotic shake-off. • 1% SDS RIPA lysis promotes robust denaturation ahead of SUMO-specific immunoprecipitation. • Compatible with downstream readouts such as western blots and mass spectrometry.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5648"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067152/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679395","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":"ELISA-Based Enzyme Kinetics Assay for Measuring cGAS Activity.","authors":"Cécile Fréreux, Philip H Howe","doi":"10.21769/BioProtoc.5655","DOIUrl":"https://doi.org/10.21769/BioProtoc.5655","url":null,"abstract":"<p><p>Cyclic GMP-AMP synthase (cGAS) is a key cytosolic double-stranded DNA sensor that activates innate immune responses. Upon binding double-stranded DNA, cGAS undergoes conformational activation and catalyzes the synthesis of the second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP) from ATP and GTP. 2'3'-cGAMP then triggers a downstream signaling cascade that induces type-I interferon and inflammatory gene expression and has been shown to exert antitumor effects in the context of cancer. Accurate measurement of this enzymatic activity is therefore important for mechanistic studies. Traditional kinetic methods such as radiolabeling, HPLC, or mass spectrometry provide precise results but require specialized equipment and expertise. Here, we describe a rapid and accessible ELISA-based protocol to quantify 2'3'-cGAMP product formation and derive cGAS enzymatic parameters. Reactions are initiated with defined DNA ligands and quenched at multiple time points, and product accumulation is quantified by a commercially available 2'3'-cGAMP ELISA. Time course measurements are used to calculate initial velocities, which can be plotted against substrate concentration to obtain Michaelis-Menten parameters. This approach enables direct, product-specific quantification of 2'3'-cGAMP formation using only an absorbance plate reader. The protocol provides a sensitive and broadly applicable alternative to traditional methods, allowing laboratories without advanced instrumentation to perform reliable cGAS enzyme kinetics. Key features • Developed to evaluate how cofactors or regulatory proteins modulate cGAS activity in vitro. • Suitable for assessing cGAS kinetics with diverse nucleic acid substrates under defined reaction conditions. • Determination of initial (V₀) and maximal (V_max) reaction rates, catalytic turnover number (k_cat), and k_1/2, the DNA concentration at which cGAS reaches 1/2 V_max. • Yields reproducible kinetic results within ~4 h from reaction setup to data analysis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5655"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067154/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679448","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":"3D STED Super-Resolution Imaging Strategy for Visualizing Synaptic Nano-architecture in Brain Cryosections.","authors":"James Scripter, Adam Skeens, Grace Jones, Yeasmin Akter, Martin Hruska","doi":"10.21769/BioProtoc.5644","DOIUrl":"10.21769/BioProtoc.5644","url":null,"abstract":"<p><p>Super-resolution imaging of synapses in intact brain tissue remains challenging because light scattering, photobleaching, and limited probe penetration, along with antigen accessibility within the densely packed postsynaptic densities (PSDs), constrain resolution and labeling efficiency. Here, we present a protocol utilizing thin brain cryosections and tau-stimulated emission depletion (STED) nanoscopy to visualize the intricate nano-architecture of excitatory synapses in situ. Slicing the brain into 6 μm sections allows for highly efficient and even penetration of probes throughout sections while ensuring that the resolution is not significantly impacted by the imaging depth of the tissue. We outline step-by-step instructions for labeling pre- and postsynaptic nano-architecture using antibodies and nanobodies, highlighting how fixative choice influences the labeling efficiency of synaptic proteins. While this protocol is compatible with both confocal and super-resolution imaging, when combined with rapid image acquisition times of tau-STED, it enables clear separation of key synaptic features in three dimensions with minimal photobleaching. Thus, this approach enables robust multiplex imaging of fluorescently labeled synaptic proteins in the brain, providing exceptional spatial resolution for visualization and quantification of synaptic nanoarchitecture in its native environment. Key features • Detailed protocol for in situ 3D STED microscopy with ~50 nm XY and ~100 nm Z resolution. • Optimized strategies for labeling pre- and postsynaptic nano-architecture using antibodies and nanobodies, including guidance on fixative choice. • Unified workflow for visualizing synaptic morphology and nanoarchitecture to uncover molecular synaptic diversity in the brain at the nanoscale.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 7","pages":"e5644"},"PeriodicalIF":1.1,"publicationDate":"2026-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13067153/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147679399","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}