Bio-protocol最新文献

{"title":"Tracking Oral Nanoparticle Uptake in Mouse Gastrointestinal Tract by Fluorescent Labeling and t-SNE Flow Cytometry.","authors":"Rabeya Jafrin Mow, Michal Pawel Kuczma, Xiaodi Shi, Didier Merlin, Chunhua Yang","doi":"10.21769/BioProtoc.5309","DOIUrl":"10.21769/BioProtoc.5309","url":null,"abstract":"<p><p>The growing demand for advanced analytical techniques to explore complex cellular targets of nanotherapeutics has driven the development of innovative methodologies. This protocol presents a refined approach for fluorescent labeling and flow cytometric analysis of colonic cells following oral lipid nanoparticle (LNP) treatment, focusing on LNP uptake in colonic cell subpopulations in a DSS-induced colitis mouse model. By integrating optimized fluorochrome selection and gating strategies with advanced t-distributed stochastic neighbor embedding (t-SNE) analysis, this method enables precise identification and multidimensional visualization of LNP-targeted epithelial and macrophage populations under the complex conditions of inflamed colon tissue. Building on our previous studies demonstrating the effectiveness of nanoparticles in targeted drug delivery, this approach highlights the utility of flow cytometry for assessing uptake efficiency and cellular targeting. Unlike conventional protocols, it incorporates t-SNE for enhanced multidimensional analysis, allowing for the detection of subtle cellular patterns and the delineation of intricate clusters. By addressing gaps in traditional methodologies, this protocol provides a robust and reproducible framework for investigating in vivo cellular targets and optimizing drug delivery strategies for nanomedicines. Key features • This protocol is optimized for investigating nanoparticle uptake in inflamed colonic tissues from DSS-induced colitis models. • This protocol integrates flow cytometry with t-SNE for high-dimensional data analysis, enabling detailed characterization of cellular populations.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5309"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104837/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164289","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":"Electrophysiological Evaluation of a Sciatic Nerve Degree III Injury Model in Rats.","authors":"Linyu Chen, Ziyi Wang, Chengyao Wang, Ning Ma, Weiqin Zhao, Shuang Liu, Jiajun Lu, Boyuan Zhao, Hong Sun, Pengcheng Che, Na Dou","doi":"10.21769/BioProtoc.5311","DOIUrl":"10.21769/BioProtoc.5311","url":null,"abstract":"<p><p>Sciatic nerve injury is a prevalent traumatic condition that significantly impacts a patient's quality of life. The sciatic nerve compression injury model is among the most commonly utilized models for investigating nerve repair and regeneration. Within this context, the degree III sciatic nerve injury model is frequently employed in scientific research due to its clinical relevance and its suitability for studies focused on functional recovery. However, a standardized approach for accurately assessing the success of constructing the degree III sciatic nerve injury model remains lacking. Traditional macroscopic observation methods exhibit limitations, whereas neurophysiological testing serves as a highly sensitive and objective evaluation technique that can directly reflect changes in nerve conduction function, thus providing reliable quantitative evidence for the successful establishment of the model. This study aims to offer a comprehensive description of the application of neurophysiological techniques in evaluating the construction of the degree III sciatic nerve injury model, thereby ensuring the success of model preparation. Key features • Precisely simulate a degree III sciatic nerve injury. • Conduct neurophysiological tests on the sciatic nerve 30 min after the injury. • Use neurophysiological techniques to improve the accuracy and repeatability of scientific study results.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5311"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164013","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":"Synchronized Visualization and Analysis of Intracellular Trafficking and Maturation of <i>Orthoflavivirus</i> Subviral Particles.","authors":"Kotaro Ishida, Eiji Morita","doi":"10.21769/BioProtoc.5324","DOIUrl":"10.21769/BioProtoc.5324","url":null,"abstract":"<p><p><i>Orthoflavivirus</i> is an enveloped, positive-stranded RNA virus that buds into the endoplasmic reticulum (ER) lumen. The budded virus particles are subsequently transported to the Golgi apparatus and secreted into the extracellular environment via the conventional secretion pathway. In this protocol, we describe a method for monitoring the secretion of <i>Orthoflavivirus</i> particles from the ER. To visualize intracellular membrane trafficking, we combine two distinct imaging techniques: the retention using selective hooks (RUSH) system and the split green fluorescent protein (GFP) system. In this approach, GFP11, a peptide tag fused to prME, the outer coat structural protein of Japanese encephalitis virus particles, was co-expressed in HeLa cells along with two additional components: GFP1-10 fused to a streptavidin-binding peptide and a <i>hook</i> construct consisting of streptavidin fused to the ER retention sequence KDEL. Time-lapse imaging was performed after the addition of biotin, which releases the captured GFP-labeled subviral particles from the ER. This method enables synchronized visualization of intracellular subviral particle trafficking and serves as a valuable tool for analyzing the maturation process of <i>Orthoflavivirus</i> particles within cells. Key features • Synchronized intracellular movement of <i>Orthoflavivirus</i> particles is visualized by the retention using selective hooks (RUSH) system. • Split GFP system is used to label viral particles. • This protocol has broader applications in investigating the transport of secretory proteins, especially those that are challenging to tag with full-length fluorescent proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5324"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104876/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164285","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 Miniprep of Intact Chloroplasts from <i>Arabidopsis thaliana</i> Leaves.","authors":"Brenda A Carranza-Correa, Karen M Dahlman-Cabrera, Manuel Gutiérrez-Aguilar","doi":"10.21769/BioProtoc.5313","DOIUrl":"10.21769/BioProtoc.5313","url":null,"abstract":"<p><p>Cell subfractionation is a common technique employed in many research laboratories to isolate organelles or intracellular compartments for the study of metabolism or biomolecule purification. While numerous protocols exist for isolating organelles, few are specifically designed for starting materials in the milligram range. Here, we present a detailed milligram-scale miniprep protocol for purifying intact chloroplasts from <i>Arabidopsis thaliana</i> leaves. This chloroplast miniprep procedure is suitable for applications such as confocal microscopy, western blotting, enzymatic assays, and other downstream analyses. Key features • This protocol condenses key aspects of existing methods and adapts them to the miniprep scale. • Researchers can isolate purified chloroplasts within 1.5 h. • This protocol requires standard laboratory equipment, such as microcentrifuges (ultracentrifuges are not required). Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5313"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104829/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164158","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":"Dissection and Whole-Mount Immunofluorescent Staining of Mouse Hind Paw Muscles for Neuromuscular Junction Analysis.","authors":"Rebecca L Simkin, Elena R Rhymes, Qiuhan Lang, Nicol Birsa, James N Sleigh","doi":"10.21769/BioProtoc.5315","DOIUrl":"10.21769/BioProtoc.5315","url":null,"abstract":"<p><p>The neuromuscular junction (NMJ) is a peripheral synaptic connection between a lower motor neuron and skeletal muscle fibre that enables muscle contraction in response to neuronal stimulation. NMJ dysfunction and morphological abnormalities are commonly observed in neurological conditions, including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy. Employing precise and reproducible techniques to visualise NMJs in mouse models of neuromuscular disorders is crucial for uncovering aspects of neuropathology, revealing disease mechanisms, and evaluating therapeutic approaches. Here, we present a method for dissecting the deep lumbrical and flexor digitorum brevis (FDB) muscles of the mouse hind paw and describe the process of whole-mount immunofluorescent staining for morphological analysis of NMJs. Similar whole-mount techniques have been applied to other muscles, such as the diaphragm; however, dense connective tissue in adult samples often impedes antibody penetration. Moreover, large hind limb muscles, including the gastrocnemius and tibialis anterior, are commonly used to examine NMJs but require embedding and cryosectioning. These additional steps increase the complexity and duration of the protocol and can introduce sectioning artefacts, including transection of NMJs and disruption of morphology. Using small hind paw muscles enables whole-mounting, which completely eliminates the requirement for embedding and cryosectioning. As a result, the entire neuromuscular innervation pattern can be visualised, allowing a more accurate assessment of NMJ development, denervation, and regeneration in mouse models of neurological disease and nerve injury, which can be applied across all postnatal ages. Key features • Small muscles of the mouse hind paw, i.e., lumbrical and FDB muscles, can be rapidly dissected for whole-mount immunofluorescent analysis without the need for cryosectioning. • This protocol allows visualisation of the entire neuromuscular innervation pattern using axonal (anti-tubulin βIII), pre-synaptic (anti-synaptophysin), and post-synaptic (α-bungarotoxin) markers. • Whole-mount immunofluorescence of hind paw muscles enables assessment of developmental, degenerative, and regenerative phenotypes in young and adult mice across disease and injury models. • High-throughput analysis can be performed using NMJ-Analyser or NMJ-morph to evaluate diverse morphological features of the NMJ.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5315"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104838/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164072","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":"ChIP-seq Data Processing and Relative and Quantitative Signal Normalization for <i>Saccharomyces cerevisiae</i>.","authors":"Kris G Alavattam, Bradley M Dickson, Rina Hirano, Rachel Dell, Toshio Tsukiyama","doi":"10.21769/BioProtoc.5299","DOIUrl":"10.21769/BioProtoc.5299","url":null,"abstract":"<p><p>Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) is a widely used technique for genome-wide analyses of protein-DNA interactions. This protocol provides a guide to ChIP-seq data processing in <i>Saccharomyces cerevisiae</i>, with a focus on signal normalization to address data biases and enable meaningful comparisons within and between samples. Designed for researchers with minimal bioinformatics experience, it includes practical overviews and refers to scripting examples for key tasks, such as configuring computational environments, trimming and aligning reads, processing alignments, and visualizing signals. This protocol employs the <b>s</b>ans-spike-<b>i</b>n method for <b>q</b>uantitative <b>ChIP</b>-seq (siQ-ChIP) and normalized coverage for absolute and relative comparisons of ChIP-seq data, respectively. While spike-in normalization, which is semiquantitative, is addressed for context, siQ-ChIP and normalized coverage are recommended as mathematically rigorous and reliable alternatives. Key features • ChIP-seq data processing workflow for Linux and macOS integrating data acquisition, trimming, alignment, processing, and multiple forms of signal computation, with a focus on reproducibility. • ChIP-seq signal generation using siQ-ChIP to quantify absolute IP efficiency-providing a rigorous alternative to spike-in normalization-and normalized coverage for relative comparisons. • Broad applicability demonstrated with <i>Saccharomyces cerevisiae</i> (experimental) and <i>Schizosaccharomyces pombe</i> (spike-in) data but suitable for ChIP-seq in any species. • In-depth notes and troubleshooting guide users through setup challenges and key concepts in basic bioinformatics, data processing, and signal computation. Graphical overview Flowchart depicting ChIP-seq data processing steps covered in this protocol.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5299"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067309/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144014378","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 Guide to Basic RNA Sequencing Data Processing and Transcriptomic Analysis.","authors":"Rowayna Shouib, Gary Eitzen, Rineke Steenbergen","doi":"10.21769/BioProtoc.5295","DOIUrl":"10.21769/BioProtoc.5295","url":null,"abstract":"<p><p>RNA sequencing (RNA-Seq) has transformed transcriptomic research, enabling researchers to perform large-scale inspection of mRNA levels in living cells. With the growing applicability of this technique to many scientific investigations, the analysis of next-generation sequencing (NGS) data becomes an important yet challenging task, especially for researchers without a bioinformatics background. This protocol offers a beginner-friendly step-by-step guide to analyze NGS data (starting from raw .fastq files), providing the required codes with an explanation of the different steps and software used. We outline a computational workflow that includes quality control, trimming of reads, read alignment to the genome, and gene quantification, ultimately enabling researchers to identify differentially expressed genes and gain insights on mRNA levels. Multiple approaches to visualize this data using statistical and graphical tools in R are also described, allowing the generation of heatmaps and volcano plots to represent genes and gene sets of interest. Key features • Provides a beginner-friendly protocol for RNA-Seq analysis to obtain insights into gene expression. • Pipeline starts with raw .fastq files and involves analysis in command line/terminal and R (via RStudio). • Yields a variety of output files that represent mRNA levels amongst different samples. Output files include count files, heatmaps, ordered lists of DEGs, and volcano plots.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5295"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067304/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144014263","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":"Optimized Protocol for DNA Extraction in Three <i>Theobroma</i> Species.","authors":"Angie F Riascos-España, Brayan Toro A Cuastumal, María Castro I Zambrano, Juan Zambrano C Arteaga, Pedro A Velasquez-Vasconez","doi":"10.21769/BioProtoc.5297","DOIUrl":"10.21769/BioProtoc.5297","url":null,"abstract":"<p><p>DNA extraction is a crucial step in molecular biology research, particularly for genetic and genomic analyses. These studies require a high concentration of high-quality DNA, which is often a challenge for underexplored species or when the available plant material consists of aged tissue. To address these challenges, the cetyltrimethylammonium bromide (CTAB)-based DNA extraction method has been optimized to improve efficiency and yield. The process begins with an overnight incubation of plant tissue macerated with liquid nitrogen in a solution containing a high concentration of CTAB (4%). Subsequently, the mixture undergoes two washes with chloroform: isoamyl alcohol. The nucleic acids are then precipitated using isopropanol, followed by a wash with 70% ethanol to ensure purity. Finally, the purified DNA is resuspended in ultrapure water. This optimized procedure produces high-quality DNA suitable for various downstream applications, including PCR and sequencing, even from older leaves of the three <i>Theobroma</i> species: <i>T. cacao, T. bicolor</i>, and <i>T. grandiflorum</i>. Additionally, this protocol significantly enhances throughput and allows for the parallel processing of a substantially larger number of samples compared to conventional techniques. Key features • An efficient CTAB-based DNA extraction protocol provides high-quality nucleic acids from older leaves by increasing CTAB concentration and incubation time in lysis buffer. • Provides reliable yields in the three <i>Theobroma</i> species: <i>T. cacao, T. bicolor</i>, and <i>T. grandiflorum</i>. • A high-throughput workflow reduces processing time and increases daily sample capacity, supporting large-scale genomic investigations.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5297"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067297/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144014320","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":"snPATHO-seq: A Detailed Protocol for Single Nucleus RNA Sequencing From FFPE.","authors":"Wani Arjumand, Kellie Wise, Hannah DuBose, Jasmine T Plummer, Luciano G Martelotto","doi":"10.21769/BioProtoc.5291","DOIUrl":"10.21769/BioProtoc.5291","url":null,"abstract":"<p><p>Formalin-fixed paraffin-embedded (FFPE) samples remain an underutilized resource in single-cell omics due to RNA degradation from formalin fixation. Here, we present snPATHO-seq, a robust and adaptable approach that enables the generation of high-quality single-nucleus (sn) transcriptomic data from FFPE tissues, utilizing advancements in single-cell genomic techniques. The snPATHO-seq workflow integrates optimized nuclei isolation with the 10× Genomics Flex assay, targeting short RNA fragments to mitigate FFPE-related RNA degradation. Benchmarking against standard 10× 3' and Flex assays for fresh/frozen tissues confirmed robust detection of transcriptomic signatures and cell types. snPATHO-seq demonstrated high performance across diverse FFPE samples, including diseased tissues like breast cancer. It seamlessly integrates with FFPE spatial transcriptomics (e.g., FFPE Visium) for multi-modal spatial and single-nucleus profiling. Compared to workflows like 10× Genomics' snFFPE, snPATHO-seq delivers superior data quality by reducing tissue debris and preserving RNA integrity via nuclei isolation. This cost-effective workflow enables high-resolution transcriptomics of archival FFPE samples, advancing single-cell omics in translational and clinical research. Key features • Optimized nuclei isolation from FFPE tissues enables high-quality single-nucleus transcriptomics by minimizing debris and maximizing intact nuclear yield. • Compatible with 10× Genomics Flex, leveraging short RNA probes to overcome FFPE RNA fragmentation challenges. • Outperforms existing FFPE workflows in cell type detection sensitivity across archival, degraded, or aged samples. • Low-cost, accessible protocol using off-the-shelf reagents, suitable for broad translational and archival tissue applications.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5291"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067313/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144058904","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":"Standardized Flow Cytometry Method for Absolute Counting of Intraepithelial Lymphocytes in the Intestinal Mucosa Using TruCount^TM Beads.","authors":"Corentin Joulain, Stéphanie Bessoles, Andrada S Chiron, Guillaume Sarrabayrouse, Salima Hacein-Bey-Abina","doi":"10.21769/BioProtoc.5307","DOIUrl":"10.21769/BioProtoc.5307","url":null,"abstract":"<p><p>In the intestinal epithelium, intraepithelial lymphocytes (IELs) coexist with intestinal epithelial cells (IECs). The IELs have an important role in defending the intestinal tract against pathogens and eliminating tumor cells. Anomalies in the absolute IEL count have been reported in various digestive diseases. IELs are typically counted using histologic techniques or under light microscopy after isolation of the epithelium. However, these techniques can introduce bias, which might account for the discrepancies in counts from one study to another. Here, we describe a flow cytometry assay for determining the absolute IEL count and the IEL/IEC ratio. We combined a conventional epithelial isolation method with a BD TruCount^TM bead-based absolute counting technique to quantify IELs (CD45⁺ CD326/EpCAM- CD103⁺CD3⁺) and IECs (CD45- CD326/EpCAM⁺) in a C57BL/6 mouse model. Key features • Intraepithelial lymphocytes (IELs) play a crucial role in maintaining mucosal integrity and defending against pathogens. • Conventional manual counting of IELs using a hemocytometer relies heavily on the operator's expertise. • Flow cytometry offers a more standardized approach to cell counting. • Using TruCount^TM beads to quantify IELs and intraepithelial cells (IECs) by flow cytometry and assess their ratio ensures reproducibility and comparison with immunohistochemical methods. Graphical Overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5307"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12089825/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144113046","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}