Bio-protocol最新文献

{"title":"Assembly and Mutagenesis of Human Coronavirus OC43 Genomes in Yeast via Transformation-Associated Recombination.","authors":"Brett A Duguay, Craig McCormick","doi":"10.21769/BioProtoc.5422","DOIUrl":"10.21769/BioProtoc.5422","url":null,"abstract":"<p><p>Human coronavirus OC43 (HCoV-OC43) is an endemic \"common cold\" coronavirus widely used to study fundamental aspects of coronavirus biology and to test therapeutic interventions. Recently, we used a yeast-based reverse genetics strategy to create recombinant HCoV-OC43 and fluorescent reporter viruses. We assembled a DNA copy of the HCoV-OC43 genome from six linear dsDNA fragments and a linearized yeast centromeric plasmid/bacterial artificial chromosome (YCpBAC) vector in <i>Saccharomyces cerevisiae</i> using transformation-associated recombination (TAR). Reporter genes encoding mCardinal fluorescent protein or histone H2B fused to mClover3 (<i>mClover-H2B</i>) or mRuby3 (<i>mRuby-H2B</i>) were inserted into an intergenic region between the HCoV-OC43 <i>M</i> and <i>N</i> genes. Assembled full-length HCoV-OC43-encoding plasmids were delivered into permissive mammalian cells to initiate viral gene expression, genome replication, and production of infectious progeny. This technique allows for the precise mutagenesis of any area of the HCoV-OC43 genome using homologous recombination, yielding genetically defined reference plasmids for the future generation of HCoV-OC43 virus stocks. Key features • Utilizes the previously developed TAR assembly method [1] to assemble and mutagenize a double-stranded DNA copy of the single-stranded RNA HCoV-OC43 genome [2]. • The availability of multiple sub-genomic and full-length HCoV-OC43-encoding plasmids provides flexibility in how substitution or deletion mutations can be incorporated using PCR or restriction cloning. • Reporter viruses enable rapid visualization and quantification of infection. • Generate and isolate a mutagenized HCoV-OC43 plasmid in approximately 14 days, followed by rescue of infectious virus in an additional 12-16 days.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5422"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378416/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982326","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":"An Ex Vivo Protocol to Assess IRE1α-Dependent RNA Cleavage Using Total RNA Isolated from Mouse Tissues.","authors":"Mohammad Mamun Ur Rashid, So-Young Rah, Se-Woong Ko, Han-Jung Chae","doi":"10.21769/BioProtoc.5414","DOIUrl":"10.21769/BioProtoc.5414","url":null,"abstract":"<p><p>Regulated IRE1-dependent decay (RIDD) is a critical cellular mechanism mediated by the endoplasmic reticulum (ER) stress sensor IRE1α, which cleaves a variety of RNA targets to regulate ER homeostasis. Current in vitro assays to study IRE1α activity largely rely on synthetic or in vitro transcribed RNA substrates, which may not fully replicate the physiological complexities of native RNA molecules. Here, we present a comprehensive protocol to assess IRE1α-dependent RNA cleavage activity using total RNA isolated directly from mouse tissues. This protocol provides a step-by-step guide for tissue collection, RNA isolation, an ex vivo RIDD assay, cDNA synthesis, and subsequent RT-PCR analysis of target mRNA cleavage products. Key reagents include active IRE1α protein, the RIDD-specific inhibitor 4μ8C, and target-specific primers for RIDD-regulated genes such as <i>Bloc1s1</i> and <i>Col6a1</i>. Quantitative assessment is achieved using agarose gel electrophoresis and imaging software. This methodology enables the study of IRE1α's RNA cleavage activity under conditions that closely mimic in vivo environments, providing a more physiologically relevant approach to understanding the role of RIDD in cellular and tissue-specific contexts. Key features • Uses total RNA from mouse tissues instead of synthetic RNA to better reflect in vivo conditions. • Includes RIDD-specific controls such as IRE1α inhibitor (4μ8C) and RNase A to confirm targeted RNA cleavage. • Combines agarose gel electrophoresis and ImageJ quantification for both qualitative and statistical validation. • Allows comparative studies of IRE1α activity across multiple mouse tissues in different biological contexts.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5414"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12399364/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144994721","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":"Protocol for Quantifying γH2AX Foci in Irradiated Cells Using Immunofluorescence and Fiji Software.","authors":"Lu Deng, Danning Wang, Lingying Wu","doi":"10.21769/BioProtoc.5421","DOIUrl":"10.21769/BioProtoc.5421","url":null,"abstract":"<p><p>Quantification of DNA double-strand breaks (DSBs) is critical for assessing genomic damage and cellular response to stress. γH2AX is a well-established marker for DNA double-strand breaks, but its quantification is often performed manually or semi-quantitatively, lacking standardization and reproducibility. Here, we present a standardized and automated workflow for γH2AX foci quantification in irradiated cells using immunofluorescence and a custom Fiji macro. The protocol includes steps for cell irradiation, immunostaining, image acquisition, and automated foci counting. The protocol is also adaptable to colony-like formations in multi-well plates, extending its utility to clonogenic assays. This protocol enables high-throughput, reproducible quantification of DNA damage with minimal user bias and can be readily implemented in routine laboratory settings. Key features • Provides an automated Fiji macro for high-throughput quantification of nuclear γH2AX fluorescence foci with single-nucleus resolution. • Standardized workflow optimized for reproducibility and cross-sample consistency in DSBs detection. • Applicable to nuclear fluorescence foci, as well as colony-like structures in multi-well formats for DNA damage and clonogenic assays.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5421"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378418/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982254","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":"Integrated Membrane Yeast Two-Hybrid System for the Analysis of Membrane Protein Complexes.","authors":"Brianna L Greenwood, Kana Oshima, David T Stuart","doi":"10.21769/BioProtoc.5418","DOIUrl":"10.21769/BioProtoc.5418","url":null,"abstract":"<p><p>Protein-protein interactions facilitate cellular functions through the creation of networks and multi-protein complexes. Mapping the interactions within and between protein networks and elucidating the composition of protein complexes provides critical insight into biological processes. Interactions among soluble cytoplasmic proteins have been extensively investigated through the application of immunoaffinity capture as well as conventional nuclear two-hybrid testing. The integrated membrane yeast two-hybrid provides a method to investigate protein-protein interactions between integral membrane proteins in their native membrane environment. This procedure makes use of the ability of the amino-terminal fragment of ubiquitin (Nub) and the carboxyl-terminal fragment of ubiquitin (Cub) to refold reconstituting functional ubiquitin, which can be recognized by a ubiquitin peptidase. Appending a fusion protein composed of Cub fused to LexA and VP16 (CLV) to a candidate \"bait\" protein and Nub to candidate \"prey\" proteins allows a test of their interaction. If the two proteins interact closely, the CLV fragment is cleaved and enters the nucleus to activate the expression of reporter genes, signaling the interaction. When the bait and prey proteins are tagged with CLV and NubG, respectively, at their genomic loci, they are only copies of the bait and prey in the cell and are expressed under the regulation of their native promoters. This avoids overexpression artifacts that can occur if the tagged proteins are expressed from plasmids while the untagged chromosomally encoded copies of the bait and prey continue to be expressed. Key features • Allows an in vivo interaction test with integral membrane proteins in the native membrane environment. • Allows integration of NubG tag at the amino or carboxyl-terminus of prey proteins. • Avoids overexpression artifacts that can be caused by expression of CLV-tagged bait and NubG-tagged prey proteins from plasmid-based systems. • Avoids competition from untagged chromosomally encoded bait and prey proteins, as occurs when CLV-tagged bait and NubG-tagged prey are expressed from plasmids.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5418"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378420/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982272","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":"Prokaryotic Expression and Purification of the hSox2-HMG Domain.","authors":"Lijie Yang, Wansen Tan, Jingjun Hong","doi":"10.21769/BioProtoc.5394","DOIUrl":"10.21769/BioProtoc.5394","url":null,"abstract":"<p><p>The Sox (SRY-related HMG-box) protein family plays a crucial role in cellular differentiation, development, and gene regulation, with the HMG (high-mobility group) domain responsible for DNA binding and transcriptional regulation. Proteins in the SOX gene family contain an HMG domain that shares 50% homology with the HMG domain of the sex-determining factor SRY gene. The SOX gene family comprises 30 proteins, which are classified into 10 groups (A-H). As a member of this family, hSox2 has been shown to be involved in various biological processes, but its specific function remains unclear. Previous studies have used eukaryotic expression systems, GST-tag purification, and bacterial inclusion body refolding techniques to produce Sox family proteins. However, these methods are often limited by issues such as low yield, incorrect folding, or inefficient purification, restricting their application in functional and structural studies. In this study, a prokaryotic expression system for the hSox2-HMG domain was constructed using the pET22b vector and <i>Escherichia coli</i> BL21(DE3) as the host strain. Protein expression was induced by IPTG, and initial purification was performed using Ni-NTA affinity chromatography, followed by ultrafiltration concentration and size exclusion chromatography to improve purity. By optimizing lysis and elution conditions, we successfully obtained hSox2-HMG protein with high expression levels and purity. This method provides a cost-effective and scalable strategy for hSox2-HMG production, ensuring high purity and correct folding of the protein. The optimized experimental protocol lays a foundation for structural and functional studies of hSox2-HMG. Key features • The hSox2-HMG protein was expressed in <i>Escherichia coli</i> BL21(DE3) using the pET22b vector and IPTG induction, resulting in high-yield recombinant protein. • Ni-NTA affinity chromatography was employed for protein purification and combined with ultrafiltration concentration and size exclusion chromatography to enhance purity and ensure correct folding. • The established workflow provides an efficient, cost-effective, and scalable strategy for hSox2-HMG production, suitable for structural and functional studies. • The purified hSox2-HMG protein maintains structural integrity and can be used for further investigation of DNA-binding properties and regulatory functions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5394"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378428/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982290","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":"An Optimized Enzyme-Coupled Spectrophotometric Method for Measuring Pyruvate Kinase Kinetics.","authors":"Saurabh Upadhyay","doi":"10.21769/BioProtoc.5415","DOIUrl":"10.21769/BioProtoc.5415","url":null,"abstract":"<p><p>Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme that catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, producing ATP in the final step of glycolysis. Unlike other isoforms, PKM2 is uniquely regulated, shifting between active tetramers and less active dimers to balance energy production with biosynthetic demands. This flexibility is exploited in cancer cells to support the Warburg effect and anabolic growth. Additionally, PKM2 can translocate to the nucleus and act as a transcriptional co-activator, influencing gene expression and tumor progression. To facilitate functional studies of PKM2, we present a robust and reproducible protocol for its expression, purification, and enzymatic characterization. PKM2 is expressed in <i>E. coli</i> and purified via Ni-NTA affinity and size-exclusion chromatography to ensure high purity and proper folding. Enzymatic activity is measured using a lactate dehydrogenase (LDH)-coupled assay that tracks NADH oxidation at 340 nm, allowing sensitive kinetic analysis under various conditions, including different PEP concentrations, pH levels, and presence of the allosteric activator fructose-1,6-bisphosphate (FBP). This non-radioactive, high-resolution method is suitable for analyzing PKM2 regulation, post-translational modifications, and mutant variants, as well as for screening potential therapeutic modulators, providing a valuable tool for cancer metabolism research. Key features • Enables robust and scalable expression of recombinant wild-type PKM2 in <i>E. coli</i>, yielding protein suitable for biochemical and structural studies. • Utilizes a non-radioactive, LDH-coupled spectrophotometric assay to accurately measure PKM2 enzymatic activity in real time by monitoring NADH consumption at 340 nm. • Supports kinetic analysis under physiologically relevant conditions, including variable pH and in the presence or absence of the allosteric activator fructose-1,6-bisphosphate (FBP). • Suitable for comparative activity profiling of PKM2 variants, mutants, or post-translationally modified forms.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5415"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378421/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982046","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":"Intravitreal NHS-Biotin Injection and Immunohistochemistry to Label and Image Protein Transport in the Mouse Optic Nerve.","authors":"Caroline R McKeown, Lucio M Schiapparelli, Hollis T Cline","doi":"10.21769/BioProtoc.5419","DOIUrl":"10.21769/BioProtoc.5419","url":null,"abstract":"<p><p>The process of moving proteins and organelles along the axon is essential for neuronal survival and function, ensuring proper communication between the cell body and distant synapses. The efficient and precise delivery of proteins via axon transport is critical for processes ranging from synaptic plasticity and neurotransmission to neuronal growth and maintenance. However, the identities of all the transported proteins have only recently begun to be investigated. Retinal ganglion cells (RGCs) provide a unique opportunity for access to central nervous system (CNS) axons as the retina is located outside the brain in the eye, with long axonal projections (~1 cm in mouse) that innervate the brain. We have developed and optimized methods for unbiased in vivo protein labeling in rodent RGC somata with intravitreal <i>N</i>-hydroxysuccinimido (NHS)-biotin and subsequent visualization of transported proteins along the optic nerve using confocal microscopy. Here, we describe these procedures in detail. Key features • Expands on the intravitreal injection method presented in Schiapparelli et al. [1] and extends it to whole mount optic nerve immunolabeling. • Unbiased in vivo labeling of proteins in the mouse central nervous system. • Optimized mouse intravitreal injection procedure using glass needles to reduce injury and costs. • Immunofluorescent imaging of the cleared intact optic nerve to visualize transported labeled proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5419"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378432/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982258","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":"Establishing and Maintaining 3D Organoid Cultures From Human Fallopian Tube Epithelium.","authors":"David Holthaus, Nina Hedemann, Sophia Theresa Geweniger, Huy Duc Le, Ibrahim Alkatout, Mirjana Kessler, Thomas F Meyer","doi":"10.21769/BioProtoc.5412","DOIUrl":"10.21769/BioProtoc.5412","url":null,"abstract":"<p><p>The female reproductive tract is comprised of different regions, each with distinctive physiological characteristics. One of them is the fallopian tubes, which are vital for human reproductive health and success. The ability to model their function and physiology is of utmost importance. So far, in vitro models have been based on a few immortalized or cancer cell lines derived from fallopian tube cells that lacked differentiated, specialized cell types and did not allow for the study of cancer initiation due to their implicit biases. Organoids, in contrast, overcome these limitations and provide an advanced, three-dimensional system for the study of healthy fallopian tube physiology and pathology. Fallopian tube organoids are comprised of epithelial progenitors that can be enriched using chemical or hormonal treatment into the different cell types that are found in the in vivo tissue, namely detyrosinated-tubulin-positive ciliated cells or paired-box protein 8 (PAX8)-positive secretory cells. This protocol provides a step-by-step guide for the establishment and maintenance of a long-term culture of organoids from healthy human fallopian tube tissue. The organoid model described here closely mimics the in vivo physiology and anatomy of human fallopian tube epithelium and provides a comprehensive basis for future studies on its underlying molecular characteristics and possible pathology. Key features • Provides a step-by-step guide for the establishment of long-term fallopian tube organoid cultures. • Allows for rapid extension of fallopian tube epithelial progenitor cells with a yield of up to 1 × 10⁸ cells within 3 weeks of isolation. • Fallopian tube organoids closely mimic healthy physiology, being comprised of multiple different cell types, like detyrosinated-tubulin-positive ciliated cells or paired-box protein 8 (PAX8)-positive secretory cells. • Further enrichment of secretory cells by hormonal treatment and ciliated cells by chemical treatment is possible.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 16","pages":"e5412"},"PeriodicalIF":1.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378425/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144982309","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":"Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution.","authors":"Lorena Alamillo, Alexander Black, Maggie P Y Lam, Edward Lau","doi":"10.21769/BioProtoc.5409","DOIUrl":"10.21769/BioProtoc.5409","url":null,"abstract":"<p><p>Protein synthesis and degradation (i.e., turnover) forms an important part of protein homeostasis and has been implicated in many age-associated diseases. Different cellular locations, such as organelles and membraneless compartments, often contain individual protein quality control and degradation machineries. Conventional methods to assess protein turnover across subcellular compartments require targeted genetic manipulation or isolation of specific organelles. Here we describe a protocol for simultaneous proteome localization and turnover (SPLAT) analysis, which combines protein turnover measurements with unbiased subcellular spatial proteomics to measure compartment-specific protein turnover rates on a proteome-wide scale. This protocol utilizes dynamic stable isotope labeling of amino acids in cell culture (dynamic SILAC) to resolve the temporal information of protein turnover and multi-step differential ultracentrifugation to assign proteins to multiple subcellular localizations. We further incorporate 2D liquid chromatography fractionation to greatly increase analytical depth while multiplexing with tandem mass tags (TMT) to reduce acquisition time 10-fold. This protocol resolves the spatial and temporal distributions of proteins and can also reveal temporally distinct spatial localizations within a protein pool. Key features • Captures protein turnover rates and subcellular localization of proteins. • Hyperplexing of dynamic SILAC and TMT LOPIT-DC in MS1 and MS2 level data. • Sample collection and processing can be completed within 1 week. • Allows comparison of organellar proteome turnover rates.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 15","pages":"e5409"},"PeriodicalIF":1.1,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12337001/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144839378","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 Imaging of Microvessels From Brain Tissue.","authors":"Josephine K Buff, Carolyn R Bertozzi, Tony Wyss-Coray, Sophia M Shi","doi":"10.21769/BioProtoc.5410","DOIUrl":"10.21769/BioProtoc.5410","url":null,"abstract":"<p><p>Proper brain function depends on the integrity of the blood-brain barrier (BBB), which is formed by a specialized network of microvessels in the brain. Reliable isolation of these microvessels is crucial for studying BBB composition and function in both health and disease. Here, we describe a protocol for the mechanical dissociation and density-based separation of microvessels from fresh or frozen human and murine brain tissue. The isolated microvessels retain their molecular integrity and are compatible with downstream applications, including fluorescence imaging and biochemical analyses. This method enables direct comparisons across species and disease states using the same workflow, facilitating translational research on BBB biology. Key features • The protocol employs mechanical dissociation and density-based separation to isolate microvessels from brain tissues. • The protocol was used to study molecular changes in brain microvessels in neurodegeneration and aging. • Validated downstream applications of this method include fluorescence imaging, RNA sequencing, proteomics, western blotting, and ELISA. • The protocol can be applied to fresh and frozen human and murine brain samples.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 15","pages":"e5410"},"PeriodicalIF":1.1,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12424264/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066768","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}